Methods for Diagnosing Breast Cancer Using MicroRNA Signatures

ABSTRACT

The present invention provides novel methods and compositions for the diagnosis and treatment of solid cancers. The invention also provides methods of identifying inhibitors of tumorigenesis.

GOVERNMENT SUPPORT

This invention was supported, in whole or in part, by Program ProjectGrant Nos. P01CA76259, PO₁CA81534 and P30CA56036 from the NationalCancer Institute. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Cancer, the uncontrolled growth of malignant cells, is a major healthproblem of the modern medical era and is one of the leading causes ofdeath in developed countries. In the United States, one in four deathsis caused by cancer (Jemal, A. et al., CA Cancer J. Clin. 52:23-47(2002)). Among cancers, those that arise from organs and solid tissues,known as solid cancers (e.g., colon cancer, lung cancer, breast cancer,stomach cancer, prostate cancer, pancreatic cancer) are among themost-commonly identified human cancers.

For example, prostate cancer is the most frequently diagnosednoncutaneous malignancy among men in industrialized countries, and, inthe United States, 1 in 8 men will develop prostate cancer during hislife (Simard, J. et al., Endocrinology 143(6):2029-40 (2002)). Theincidence of prostate cancer has dramatically increased over the lastdecades and prostate cancer is now a leading cause of death in theUnited States and Western Europe (Peschel, R. E. and J. W. Colberg,Lancet 4:233-41 (2003); Nelson, W. G. et al., N. Engl. J. Med.349(4):366-81 (2003)). An average 40% reduction in life expectancyaffects males with prostate cancer. If detected early, prior tometastasis and local spread beyond the capsule, prostate cancer canoften times be cured (e.g., using surgery). However, if diagnosed afterspread and metastasis from the prostate, prostate cancer is typically afatal disease with low cure rates. While prostate-specific antigen(PSA)-based screening has aided early diagnosis of prostate cancer, itis neither highly sensitive nor specific (Punglia et al., N. Engl. J.Med. 349(4):335-42 (2003)). This means that a high percentage of falsenegative and false positive diagnoses are associated with the test. Theconsequences are both many instances of missed cancers and unnecessaryfollow-up biopsies for those without cancer.

Breast cancer remains the second leading cause of cancer-related deathsin women, affecting more than 180,000 women in the United States eachyear. For women in North America, the life-time odds of getting breastcancer are now one in eight. Although the discovery of BRCA1 and BRCA2were important steps in identifying key genetic factors involved inbreast cancer, it has become clear that mutations in BRCA 1 and BRCA2account for only a fraction of inherited susceptibility to breast cancer(Nathanson, K. L., et al., Human Mol. Gen. 10(7):715-720 (2001);Anglican Breast Cancer Study Group. Br. J. Cancer 83(10):1301-08 (2000);and Syrjakoski, K., et al., J. Natl. Cancer Inst. 92:1529-31 (2000)).Despite considerable research into therapies for breast cancer, breastcancer remains difficult to diagnose and treat effectively, and the highmortality observed in breast cancer patients indicates that improvementsare needed in the diagnosis, treatment and prevention of the disease.

Excluding skin cancer, colorectal cancer is the third most frequentlydiagnosed cancer in the United States and Canada (after lung and breastin women, and lung and prostate in men). The American Cancer Societyestimates that there will be approximately 145,000 new cases ofcolorectal cancer diagnosed in the U.S. in 2005 (Cancer Facts andFigures 2005. Atlanta, Ga.: American Cancer Society, 2005. Available atwww.cancer.org/docroot/STT/stt_(—)0.asp, accessed Dec. 19, 2005).Colorectal cancer is the second leading cause of cancer death among menand women in the United States and Canada (after lung cancer).

The annual incidence of pancreatic cancer is nearly equivalent to theannual mortality, estimated to be 31,860 and 31,270, respectively, inthe U.S. in 2004 (Cancer Facts and Figures 2004. Atlanta, Ga.: AmericanCancer Society, 2004. Available atwww.cancer.org/docroot/STT/stt_(—)0_(—)2004.asp, accessed Aug. 21,2005). Patients with locally advanced and metastatic pancreatic cancerhave poor prognoses, and diagnosis generally occurs too late for surgeryor radiotherapy to be curative (Burr, H. A., et al., The Oncologist10(3): 183-190, (2005)). Chemotherapy can provide relief of symptoms forsome patients with advanced pancreatic cancer, but its impact onsurvival has been modest to date.

In the United States, more than 20,000 individuals are diagnosed withstomach (gastric) cancer each year. The American Cancer Societyestimates that there will be 22,710 new cases of colorectal cancerdiagnosed in the U.S. in 2004 (Cancer Facts and Figures 2004. Atlanta,Ga.: American Cancer Society, 2004. Available atwww.cancer.org/docroot/STT/stt_(—)0_(—)2004.asp, accessed Aug. 21,2005). Because stomach cancer may occur without symptoms, it may be inadvanced stages by the time the diagnosis is made. Treatment is thendirected at making the patient more comfortable and improving quality oflife.

Lung cancer causes more deaths worldwide than any other form of cancer(Goodman, G. E., Thorax 57:994-999 (2002)). In the United States, lungcancer is the primary cause of cancer death among both men and women. In2002, the death rate from lung cancer was an estimated 134,900 deaths,exceeding the combined total for breast, prostate and colon cancer. Id.Lung cancer is also the leading cause of cancer death in all Europeancountries, and numbers of lung cancer-related deaths are rapidlyincreasing in developing countries as well.

The five-year survival rate among all lung cancer patients, regardlessof the stage of disease at diagnosis, is only about 13%. This contrastswith a five-year survival rate of 46% among cases detected while thedisease is still localized. However, only 16% of lung cancers arediscovered before the disease has spread. Early detection is difficultas clinical symptoms are often not observed until the disease hasreached an advanced stage. Despite research into therapies for this andother cancers, lung cancer remains difficult to diagnose and treateffectively.

Clearly, the identification of markers and genes that are responsiblefor susceptibility to particular forms of solid cancer (e.g., prostatecancer, breast cancer, lung cancer, stomach cancer, colon cancer,pancreatic cancer) is one of the major challenges facing oncology today.There is a need to identify means for the early detection of individualsthat have a genetic susceptibility to cancer so that more aggressivescreening and intervention regimens may be instituted for the earlydetection and treatment of cancer. Cancer genes may also reveal keymolecular pathways that may be manipulated (e.g., using small or largemolecule weight drugs) and may lead to more effective treatmentsregardless of the cancer stage when a particular cancer is firstdiagnosed.

MicroRNAs are a class of small, non-coding RNAs that control geneexpression by hybridizing to and triggering either translationalrepression or, less frequently, degradation of a messenger RNA (mRNA)target. The discovery and study of miRNAs has revealed miRNA-mediatedgene regulatory mechanisms that play important roles in organismaldevelopment and various cellular processes, such as celldifferentiation, cell growth and cell death (Cheng, A. M., et al.,Nucleic Acids Res. 33:1290-1297 (2005)). Recent studies suggest thataberrant expression of particular miRNAs may be involved in humandiseases, such as neurological disorders (Ishizuka, A., et al., GenesDev. 16:2497-2508 (2002)) and cancer. In particular, misexpression ofmiR-16-1 and/or miR-15a has been found in human chronic lymphocyticleukemias (Calin, G. A., et al., Proc. Natl. Acad. Sci. U.S.A.99:15524-15529 (2002)).

Clearly, there is a great need in the art for improved methods fordetecting and treating solid cancers (e.g., prostate cancer, breastcancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer).The present invention provides novel methods and compositions for thediagnosis and treatment of solid cancers.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the identification ofspecific miRNAs that have altered expression levels in particular solidcancers.

Accordingly, the invention encompasses methods of diagnosing whether asubject has, or is at risk for developing, a solid cancer. According tothe methods of the invention, the level of at least one miR gene productin a test sample from the subject is compared to the level of acorresponding miR gene product in a control sample. An alteration (e.g.,an increase, a decrease) in the level of the miR gene product in thetest sample, relative to the level of a corresponding miR gene productin a control sample, is indicative of the subject either having, orbeing at risk for developing, a solid cancer. The solid cancer can beany cancer that arises from organs and solid tissues. In certainembodiments, the solid cancer is stomach cancer, breast cancer,pancreatic cancer, colon cancer, lung cancer or prostate cancer. Inparticular embodiments, the solid cancer is not breast cancer, lungcancer, prostate cancer, pancreatic cancer or gastrointestinal cancer.

In one embodiment, the at least one miR gene product measured in thetest sample is selected from the group consisting of miR-21, miR-191,miR-17-5p and combinations thereof. In another embodiment, the at leastone miR gene product measured in the test sample is selected from thegroup consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223,miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1,miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221,miR-106a and combinations thereof.

In one embodiment, the solid cancer is breast cancer or lung cancer andthe at least one miR gene product measured in the test sample isselected from the group consisting of miR-210, miR-213 and a combinationthereof.

In another embodiment, the solid cancer is colon cancer, stomach cancer,prostate cancer or pancreas cancer and the at least one miR gene productmeasured in the test sample is miR-218-2.

In a certain embodiment, the solid cancer is breast cancer and the atleast one miR gene product measured in the test sample is selected fromthe group consisting of miR-125b-1, miR-125b-2, miR-145, miR-21 andcombinations thereof. In a related embodiment, the solid cancer isbreast cancer and the at least one miR gene product in the test sampleis selected from the group consisting of miR-21, miR-29b-2, miR-146,miR-125b-2, miR-125b-1, miR-10b, miR-145, miR-181a, miR-140, miR-213,miR-29a prec, miR-181b-1, miR-199b, miR-29b-1, miR-130a, miR-155,let-7a-2, miR-205, miR-29c, miR-224, miR-100, miR-31, miR-30c,miR-17-5p, miR-210, miR-122a, miR-16-2 and combinations thereof.

In another embodiment, the solid cancer is colon cancer and the at leastone miR gene product in the test sample is selected from the groupconsisting of miR-24-1, miR-29b-2, miR-20a, miR-10a, miR-32, miR-203,miR-106a, miR-17-5p, miR-30c, miR-223, miR-126*, miR-128b, miR-21,miR-24-2, miR-99b prec, miR-155, miR-213, miR-150, miR-107, miR-191,miR-221, miR-9-3 and combinations thereof.

In yet another embodiment, the solid cancer is lung cancer and the miRgene product in the test sample is selected from the group consisting ofmiR-21, miR-205, miR-200b, miR-210, miR-148, miR-141, miR-132, miR-215,miR-128b, let-7g, miR-16-2, miR-129-1/2 prec, miR-126*, miR-142-as,miR-30d, miR-30a-5p, miR-7-2, miR-199a-1, miR-127, miR-34a prec,miR-34a, miR-136, miR-202, miR-196-2, miR-199a-2, let-7a-2, miR-124a-1,miR-149, miR-17-5p, miR-196-1 prec, miR-10a, miR-99b prec, miR-196-1,miR-199b, miR-191, miR-195, miR-155 and combinations thereof.

In an additional embodiment, the solid cancer is pancreatic cancer andthe at least one miR gene product measured in the test sample isselected from the group consisting of miR-103-1, miR-103-2, miR-155,miR-204 and combinations thereof. In a related embodiment, the solidcancer is pancreatic cancer and the miR gene product in the test sampleis selected from the group consisting of miR-103-2, miR-103-1, miR-24-2,miR-107, miR-100, miR-125b-2, miR-125b-1, miR-24-1, miR-191, miR-23a,miR-26a-1, miR-125a, miR-130a, miR-26b, miR-221, miR-126*, miR-16-2,miR-146, miR-214, miR-99b, miR-128b, miR-155, miR-29b-2, miR-29a,miR-25, miR-16-1, miR-99a, miR-224, miR-30d, miR-92-2, miR-199a-1,miR-223, miR-29c, miR-30b, miR-129-1/2, miR-197, miR-17-5p, miR-30c,miR-7-1, miR-93-1, miR-140, miR-30a-5p, miR-132, miR-181b-1, miR-152prec, miR-23b, miR-20a, miR-222, miR-27a, miR-92-1, miR-21, miR-129-1/2prec, miR-150, miR-32, miR-106a, miR-29b-1 and combinations thereof.

In another embodiment, the solid cancer is prostate cancer and the miRgene product in the test sample is selected from the group consisting oflet-7d, miR-128a prec, miR-195, miR-203, let-7a-2 prec, miR-34a,miR-20a, miR-218-2, miR-29a, miR-25, miR-95, miR-197, miR-135-2,miR-187, miR-196-1, miR-148, miR-191, miR-21, let-71, miR-198,miR-199a-2, miR-30c, miR-17-5p, miR-92-2, miR-146, miR-181b-1 prec,miR-32, miR-206, miR-184 prec, miR-29a prec, miR-29b-2, miR-149,miR-181b-1, miR-196-1 prec, miR-93-1, miR-223, miR-16-1, miR-101-1,miR-124a-1, miR-26a-1, miR-214, miR-27a, miR-24-1, miR-106a, miR-199a-1and combinations thereof.

In yet another embodiment, the solid cancer is stomach cancer and themiR gene product in the test sample is selected from the groupconsisting of miR-223, miR-21, miR-218-2, miR-103-2, miR-92-2, miR-25,miR-136, miR-191, miR-221, miR-125b-2, miR-103-1, miR-214, miR-222,miR-212 prec, miR-125b-1, miR-100, miR-107, miR-92-1, miR-96, miR-192,miR-23a, miR-215, miR-7-2, miR-138-2, miR-24-1, miR-99b, miR-33b,miR-24-2 and combinations thereof.

The level of the at least one miR gene product can be measured using avariety of techniques that are well known to those of skill in the art(e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis,solution hybridization detection). In a particular embodiment, the levelof at least one miR gene product is measured by reverse transcribing RNAfrom a test sample obtained from the subject to provide a set of targetoligodeoxynucleotides, hybridizing the target oligodeoxynucleotides toone or more miRNA-specific probe oligonucleotides (e.g., hybridzing to amicroarray that comprises several miRNA-specific probe oligonucleotides)to provide a hybridization profile for the test sample, and comparingthe test sample hybridization profile to a hybridization profile from acontrol sample. An alteration in the signal of at least one miRNA in thetest sample relative to the control sample is indicative of the subjecteither having, or being at risk for developing, a solid cancer. In aparticular embodiment, target oligonucleotides are hybridized to amicroarray comprising miRNA-specific probe oligonucleotides for one ormore miRNAs selected from the group consisting of miR-21, miR-17-5p,miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2,miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2,miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof.

The invention also encompasses methods of inhibiting tumorigenesis in asubject who has, or is suspected of having, a solid cancer (e.g.,prostate cancer, stomach cancer, pancreatic cancer, lung cancer, breastcancer, colon cancer), wherein at least one miR gene product isderegulated (e.g., down-regulated, up-regulated) in the cancer cells ofthe subject. When the at least one isolated miR gene product isdown-regulated in the cancer cells, the method comprises administeringan effective amount of an isolated miR gene product, an isolated variantor a biologically-active fragment of the miR gene product or variant,such that proliferation of cancer cells in the subject is inhibited. Ina further embodiment, the at least one isolated miR gene product isselected from the group consisting of miR-145, miR-155, miR-218-2 andcombinations thereof. In a particular embodiment, the miR gene productis not miR-15a or miR-16-1. When the at least one isolated miR geneproduct is up-regulated in the cancer cells, the method comprisesadministering to the subject an effective amount of at least onecompound for inhibiting expression of the at least one miR gene product(referred to herein as a “miR expression-inhibition compound”), suchthat proliferation of cancer cells in the subject is inhibited. In aparticular embodiment, the at least one miR expression-inhibitioncompound is specific for a miR gene product selected from the groupconsisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b,miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a,miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106aand combinations thereof.

In a related embodiment, the methods of inhibiting tumorigenesis in asubject additionally comprise the step of determining the amount of atleast one miR gene product in cancer cells from the subject, andcomparing that level of the miR gene product in the cells to the levelof a corresponding miR gene product in control cells. If expression ofthe miR gene product is deregulated (e.g., down-regulated, up-regulated)in cancer cells, the methods further comprise altering the amount of theat least one miR gene product expressed in the cancer cells. In oneembodiment, the amount of the miR gene product expressed in the cancercells is less than the amount of the miR gene product expressed in acontrol cell (e.g., control cells), and an effective amount of thedown-regulated miR gene product, isolated variant or biologically-activefragment of the miR gene product or variant, is administered to thesubject. Suitable miR gene products for this embodiment include miR-145,miR-155, miR-218-2 and combinations thereof, among others. In aparticular embodiment, the miR gene product is not miR-15a or miR-16-1.In another embodiment, the amount of the miR gene product expressed inthe cancer cells is greater than the amount of the miR gene productexpressed in the control cell (e.g., control cells), and an effectiveamount of at least one compound for inhibiting expression of the atleast one up-regulated miR gene product is administered to the subject.Suitable compounds for inhibiting expression of the at least one miRgene product include, but are not limited to, compounds that inhibit theexpression of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b,miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a,miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106aand combinations thereof.

The invention further provides pharmaceutical compositions for treatingsolid cancers (e.g., prostate cancer, stomach cancer, pancreatic cancer,lung cancer, breast cancer, colon cancer). In one embodiment, thepharmaceutical compositions comprise at least one isolated miR geneproduct and a pharmaceutically-acceptable carrier. In a particularembodiment, the at least one miR gene product corresponds to a miR geneproduct that has a decreased level of expression in cancer cellsrelative to control cells. In certain embodiments the isolated miR geneproduct is selected from the group consisting of miR-145, miR-155,miR-218-2 and combinations thereof.

In another embodiment, the pharmaceutical compositions of the inventioncomprise at least one miR expression-inhibition compound and apharmaceutically-acceptable carrier. In a particular embodiment, the atleast one miR expression-inhibition compound is specific for a miR geneproduct whose expression is greater in cancer cells than in controlcells. In certain embodiments, the miR expression-inhibition compound isspecific for one or more miR gene products selected from the groupconsisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b,miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a,miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106aand combinations thereof.

The invention also encompasses methods of identifying an inhibitor oftumorigenesis, comprising providing a test agent to a cell and measuringthe level of at least one miR gene product in the cell. In oneembodiment, the method comprises providing a test agent to a cell andmeasuring the level of at least one miR gene product associated withdecreased expression levels in solid cancers (e.g., prostate cancer,stomach cancer, pancreatic cancer, lung cancer, breast cancer, coloncancer). An increase in the level of the miR gene product in the cell,relative to a suitable control cell, is indicative of the test agentbeing an inhibitor of tumorigenesis. In a particular embodiment, the atleast one miR gene product associated with decreased expression levelsin solid cancer cells is selected from the group consisting of miR-145,miR-155, miR-218-2 and combinations thereof.

In other embodiments, the method comprises providing a test agent to acell and measuring the level of at least one miR gene product associatedwith increased expression levels in solid cancers. A decrease in thelevel of the miR gene product in the cell, relative to a suitablecontrol cell, is indicative of the test agent being an inhibitor oftumorigenesis. In a particular embodiment, the at least one miR geneproduct associated with increased expression levels in solid cancercells is selected from the group consisting of miR-21, miR-17-5p,miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2,miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-92-2, miR-214,miR-30c, miR-25, miR-221, miR-106a and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts a clustering analysis of 540 samples, representing 6solid cancers (top) and the respective normal tissues. miRNAs includedin the tree (n=137) represent those whose expression level(background-subtracted intensity) was higher than the threshold value(256) in at least 50% of the samples analyzed. Arrays weremedian-centered and normalized using Gene Cluster 2.0. Average linkageclustering was performed by using uncentered correlation metric. Thecolors indicate the difference in expression level from the median forthe microRNAs in each sample.

FIG. 2 depicts unsupervised analysis of microRNA expression data.MicroRNA profiling of 540 samples (indicated at top of panel) coveringbreast, colon, lung, pancreas, prostate and stomach (normal tissues andtumors) were filtered, centered and normalized for each feature. Thedata were subject to hierarchical clustering on both the samples(horizontally-oriented) and the features (vertically-oriented withaverage linkage and Pearson correlation as a similarity measure. Samplenames are indicated at the top of the figure and miRNA names on theleft. The probe ID is indicated in parentheses, as the same microRNA canbe measured by different oligonucleotides. The colors indicate thedifference in expression level from the median for the microRNAs in eachsample.

FIG. 3 depicts the expression of differentially-regulated miRNAs acrosssolid cancers (top). Sixty-one microRNAs, which are present in at least90% of the tissues solid cancers, are represented (right of panel). Thetree displays the average absolute expression values for each of thelisted microRNAs after log₂ transformation. The mean was computed overall samples from the same tissue or tumor histotype. Genes weremean-centered and normalized using Gene Cluster 2.0. Average linkageclustering was performed using Euclidean distance.

FIG. 4 depicts fold changes in the expression of miRNAs present in atleast 75% of the solid tumors with at least 1 tumor absolute valuehigher than 2 in different cancer samples (top), relative to normalsamples. The tree displays the log₂ transformation of average foldchanges (cancer vs. normal). The mean was computed over all samples fromthe same tissue or tumor histotype. Arrays were mean-centered andnormalized using Gene Cluster 2.0. Average linkage clustering wasperformed using uncentered correlation metric.

FIG. 5 depicts fold changes in the expression of miRNAs present in thesignatures of at least 50% of the solid tumors in cancer vs. normalsamples. The tree displays the log₂ transformation of the average foldchanges (cancer over normal). The mean was computed over all samplesfrom the same tissue or tumor histotype. Arrays were mean centered andnormalized using Gene Cluster 2.0. Average linkage clustering wasperformed using uncentered correlation metric.

FIG. 6A depicts bar graphs indicating that the 3′UTR of different genesencoding cancer protein enables cancer regulation by microRNA. Therelative repression of firefly luciferase expression (Fold Change)standardized to a renilla luciferase control. PLAG1, pleiomorphicadenoma gene 1; TGFBR2, transforming growth factor beta receptor II; Rb,retinoblastoma gene. pGL-3 (Promega) was used as the empty vector.miR-20a, miR-26a-1 and miR-106 oligoRNAs (sense and scrambled) were usedfor transfections. A second experiment using mutated versions of eachtarget mRNA, which lack the 5′ miRNA-end complementarity site (MUT), ascontrols is shown in the bottom panel. All the experiments wereperformed twice in triplicate (n=6).

FIG. 6B depicts Western blots indicating that, in certain cancers (e.g.,lung, breast, colon, gastric), the levels of RB1 (Rb) protein displaysan inverse correlation with the level of miR-106a expression. β-Actinwas used as a control for normalization. N1, normal sample; T1 and T2,tumor sample.

FIG. 7 depicts Northern blots showing down-regulation of miR-145 (top)and up-regulation of miR-21 (bottom) expression in breast cancer samples(P series and numbered series) relative to normal samples. Normalizationwas performed with a U6-specific probe.

FIG. 8 depicts Northern blots showing up-regulation of miR-103 anddown-regulation miR-155 (top) expression in different endocrinepancreatic cancer samples (WDET, well differentiated pancreaticendocrine tumors, WDEC, well differentiated pancreatic endocrinecarcinomas and ACC, pancreatic acinar cell carcinomas) relative tonormal samples (K series), as well as up-regulation of miR-204 (bottom)expression in insulinomas (F series) relative to normal samples (Kseries) and non secreting/non functioning (NF-series) samples.Normalization was performed with a probe specific to 5S RNA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the identification ofparticular microRNAs whose expression is altered in cancer cellsassociated with different solid cancers, such as colon, stomach,pancreatic, lung, breast and prostate cancer, relative to normal controlcells.

As used herein interchangeably, a “miR gene product,” “microRNA,” “miR,”or “miRNA” refers to the unprocessed (e.g., precursor) or processed(e.g., mature) RNA transcript from a miR gene. As the miR gene productsare not translated into protein, the term “miR gene products” does notinclude proteins. The unprocessed miR gene transcript is also called a“miR precursor” or “miR prec” and typically comprises an RNA transcriptof about 70-100 nucleotides in length. The miR precursor can beprocessed by digestion with an RNAse (for example, Dicer, Argonaut, orRNAse III (e.g., E. coli RNAse III)) into an active 19-25 nucleotide RNAmolecule. This active 19-25 nucleotide RNA molecule is also called the“processed” miR gene transcript or “mature” miRNA.

The active 19-25 nucleotide RNA molecule can be obtained from the miRprecursor through natural processing routes (e.g., using intact cells orcell lysates) or by synthetic processing routes (e.g., using isolatedprocessing enzymes, such as isolated Dicer, Argonaut, or RNAse III). Itis understood that the active 19-25 nucleotide RNA molecule can also beproduced directly by biological or chemical synthesis, without havingbeen processed from the miR precursor. When a microRNA is referred toherein by name, the name corresponds to both the precursor and matureforms, unless otherwise indicated.

Tables 1a and 1b depict the nucleotide sequences of particular precursorand mature human microRNAs.

TABLE 1a Human microRNA Precursor Sequences SEQ Precursor ID NameSequence (5′ To 3′)* NO. let-7a-1 CACUGUGGGAUGAGGUAGUAGGUUGUAUAGUUU 1UAGGGUCACACCCACCACUGGGAGAUAACUAUA CAAUCUACUGUCUUUCCUAACGUG let-7a-2AGGUUGAGGUAGUAGGUUGUAUAGUUUAGAAUU 2 ACAUCAAGGGAGAUAACUGUACAGCCUCCUAGCUUUCCU let-7a-3 GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUC 3UGCCCUGCUAUGGGAUAACUAUACAAUCUACUG UCUUUCCU let-7a-4GUGACUGCAUGCUCCCAGGUUGAGGUAGUAGGU 4 UGUAUAGUUUAGAAUUACACAAGGGAGAUAACUGUACAGCCUCCUAGCUUUCCUUGGGUCUUGCAC UAAACAAC let-7bGGCGGGGUGAGGUAGUAGGUUGUGUGGUUUCAG 5 GGCAGUGAUGUUGCCCCUCGGAAGAUAACUAUACAACCUACUGCCUUCCCUG let-7c GCAUCCGGGUUGAGGUAGUAGGUUGUAUGGUUU 6AGAGUUACACCCUGGGAGUUAACUGUACAACCU UCUAGCUUUCCUUGGAGC let-7dCCUAGGAAGAGGUAGUAGGUUGCAUAGUUUUAG 7 GGCAGGGAUUUUGCCCACAAGGAGGUAACUAUACGACCUGCUGCCUUUCUUAGG let-7d-v1 CUAGGAAGAGGUAGUAGUUUGCAUAGUUUUAGG 8GCAAAGAUUUUGCCCACAAGUAGUUAGCUAUAC GACCUGCAGCCUUUUGUAG let-7d-v2CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGG 9 GUUGUGACAUUGCCCGCUGUGGAGAUAACUGCGCAAGCUACUGCCUUGCUAG let-7e CCCGGGCUGAGGUAGGAGGUUGUAUAGUUGAGG 10AGGACACCCAAGGAGAUCACUAUACGGCCUCCU AGCUUUCCCCAGG let-7f-1UCAGAGUGAGGUAGUAGAUUGUAUAGUUGUGGG 11 GUAGUGAUUUUACCCUGUUCAGGAGAUAACUAUACAAUCUAUUGCCUUCCCUGA let-7f-2-1 CUGUGGGAUGAGGUAGUAGAUUGUAUAGUUGUG 12GGGUAGUGAUUUUACCCUGUUCAGGAGAUAACU AUACAAUCUAUUGCCUUCCCUGA let-7f-2-2CUGUGGGAUGAGGUAGUAGAUUGUAUAGUUUUA 13 GGGUCAUACCCCAUCUUGGAGAUAACUAUACAGUCUACUGUCUUUCCCACGG let-7g UUGCCUGAUUCCAGGCUGAGGUAGUAGUUUGUA 14CAGUUUGAGGGUCUAUGAUACCACCCGGUACAG GAGAUAACUGUACAGGCCACUGCCUUGCCAGGAACAGCGCGC let-7i CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGG 15GUUGUGACAUUGCCCGCUGUGGAGAUAACUGCG CAAGCUACUGCCUUGCUAG miR-1b-1-1ACCUACUCAGAGUACAUACUUCUUUAUGUACCC 16 AUAUGAACAUACAAUGCUAUGGAAUGUAAAGAAGUAUGUAUUUUUGGUAGGC miR-1b-1-2 CAGCUAACAACUUAGUAAUACCUACUCAGAGUA 17CAUACUUCUUUAUGUACCCAUAUGAACAUACAA UGCUAUGGAAUGUAAAGAAGUAUGUAUUUUUGGUAGGCAAUA miR-1b-2 GCCUGCUUGGGAAACAUACUUCUUUAUAUGCCC 18AUAUGGACCUGCUAAGCUAUGGAAUGUAAAGAA GUAUGUAUCUCAGGCCGGG miR-1bUGGGAAACAUACUUCUUUAUAUGCCCAUAUGGA 19 CCUGCUAAGCUAUGGAAUGUAAAGAAGUAUGUAUCUCA miR-1d ACCUACUCAGAGUACAUACUUCUUUAUGUACCC 20AUAUGAACAUACAAUGCUAUGGAAUGUAAAGAA GUAUGUAUUUUUGGUAGGC miR-7-1aUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAG 21 UGAUUUUGUUGUUUUUAGAUAACUAAAUCGACAACAAAUCACAGUCUGCCAUAUGGCACAGGCCAU GCCUCUACA miR-7-1bUUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUA 22 GUGAUUUUGUUGUUUUUAGAUAACUAAAUCGACAACAAAUCACAGUCUGCCAUAUGGCACAGGCCA UGCCUCUACAG miR-7-2CUGGAUACAGAGUGGACCGGCUGGCCCCAUCUG 23 GAAGACUAGUGAUUUUGUUGUUGUCUUACUGCGCUCAACAACAAAUCCCAGUCUACCUAAUGGUGC CAGCCAUCGCA miR-7-3AGAUUAGAGUGGCUGUGGUCUAGUGCUGUGUGG 24 AAGACUAGUGAUUUUGUUGUUCUGAUGUACUACGACAACAAGUCACAGCCGGCCUCAUAGCGCAGA CUCCCUUCGAC miR-9-1CGGGGUUGGUUGUUAUCUUUGGUUAUCUAGCUG 25 UAUGAGUGGUGUGGAGUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAAUAACCCCA miR-9-2 GGAAGCGAGUUGUUAUCUUUGGUUAUCUAGCUG 26UAUGAGUGUAUUGGUCUUCAUAAAGCUAGAUAA CCGAAAGUAAAAACUCCUUCA miR-9-3GGAGGCCCGUUUCUCUCUUUGGUUAUCUAGCUG 27 UAUGAGUGCCACAGAGCCGUCAUAAAGCUAGAUAACCGAAAGUAGAAAUGAUUCUCA miR-10a GAUCUGUCUGUCUUCUGUAUAUACCCUGUAGAU 28CCGAAUUUGUGUAAGGAAUUUUGUGGUCACAAA UUCGUAUCUAGGGGAAUAUGUAGUUGACAUAAACACUCCGCUCU miR-10b CCAGAGGUUGUAACGUUGUCUAUAUAUACCCUG 29UAGAACCGAAUUUGUGUGGUAUCCGUAUAGUCA CAGAUUCGAUUCUAGGGGAAUAUAUGGUCGAUGCAAAAACUUCA miR-15a-2 GCGCGAAUGUGUGUUUAAAAAAAAUAAAACCUU 30GGAGUAAAGUAGCAGCACAUAAUGGUUUGUGGA UUUUGAAAAGGUGCAGGCCAUAUUGUGCUGCCUCAAAAAUAC miR-15a CCUUGGAGUAAAGUAGCAGCACAUAAUGGUUUG 31UGGAUUUUGAAAAGGUGCAGGCCAUAUUGUGCU GCCUCAAAAAUACAAGG miR-15b-1CUGUAGCAGCACAUCAUGGUUUACAUGCUACAG 32 UCAAGAUGCGAAUCAUUAUUUGCUGCUCUAGmiR-15b-2 UUGAGGCCUUAAAGUACUGUAGCAGCACAUCAU 33GGUUUACAUGCUACAGUCAAGAUGCGAAUCAUU AUUUGCUGCUCUAGAAAUUUAAGGAAAUUCAUmiR-16-1 GUCAGCAGUGCCUUAGCAGCACGUAAAUAUUGG 34CGUUAAGAUUCUAAAAUUAUCUCCAGUAUUAAC UGUGCUGCUGAAGUAAGGUUGAC miR-16-2GUUCCACUCUAGCAGCACGUAAAUAUUGGCGUA 35 GUGAAAUAUAUAUUAAACACCAAUAUUACUGUGCUGCUUUAGUGUGAC miR-16-13 GCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUU 36AAGAUUCUAAAAUUAUCUCCAGUAUUAACUGUG CUGCUGAAGUAAGGU miR-17GUCAGAAUAAUGUCAAAGUGCUUACAGUGCAGG 37 UAGUGAUAUGUGCAUCUACUGCAGUGAAGGCACUUGUAGCAUUAUGGUGAC miR-18 UGUUCUAAGGUGCAUCUAGUGCAGAUAGUGAAG 38UAGAUUAGCAUCUACUGCCCUAAGUGCUCCUUC UGGCA miR-18-13UUUUUGUUCUAAGGUGCAUCUAGUGCAGAUAGU 39 GAAGUAGAUUAGCAUCUACUGCCCUAAGUGCUCCUUCUGGCAUAAGAA miR-19a GCAGUCCUCUGUUAGUUUUGCAUAGUUGCACUA 40CAAGAAGAAUGUAGUUGUGCAAAUCUAUGCAAA ACUGAUGGUGGCCUGC miR-19a-13CAGUCCUCUGUUAGUUUUGCAUAGUUGCACUAC 41 AAGAAGAAUGUAGUUGUGCAAAUCUAUGCAAAACUGAUGGUGGCCUG miR-19b-1 CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAU 42CCAGCUGUGUGAUAUUCUGCUGUGCAAAUCCAU GCAAAACUGACUGUGGUAGUG miR-19b-2ACAUUGCUACUUACAAUUAGUUUUGCAGGUUUG 43 CAUUUCAGCGUAUAUAUGUAUAUGUGGCUGUGCAAAUCCAUGCAAAACUGAUUGUGAUAAUGU miR-19b-13UUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGC 44 UGUGUGAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUAG miR-19b-X UUACAAUUAGUUUUGCAGGUUUGCAUUUCAGCG 45UAUAUAUGUAUAUGUGGCUGUGCAAAUCCAUGC AAAACUGAUUGUGAU miR-20GUAGCACUAAAGUGCUUAUAGUGCAGGUAGUGU 46 (miR-20a)UUAGUUAUCUACUGCAUUAUGAGCACUUAAAGU ACUGC miR-21UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGU 47 UGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA miR-21-17 ACCUUGUCGGGUAGCUUAUCAGACUGAUGUUGA 48CUGUUGAAUCUCAUGGCAACACCAGUCGAUGGG CUGUCUGACAUUUUG miR-22GGCUGAGCCGCAGUAGUUCUUCAGUGGCAAGCU 49 UUAUGUCCUGACCCAGCUAAAGCUGCCAGUUGAAGAACUGUUGCCCUCUGCC miR-23a GGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCU 50UCCUGUCACAAAUCACAUUGCCAGGGAUUUCCA ACCGACC miR-23bCUCAGGUGCUCUGGCUGCUUGGGUUCCUGGCAU 51 GCUGAUUUGUGACUUAAGAUUAAAAUCACAUUGCCAGGGAUUACCACGCAACCACGACCUUGGC miR-23-19CCACGGCCGGCUGGGGUUCCUGGGGAUGGGAUU 52 UGCUUCCUGUCACAAAUCACAUUGCCAGGGAUUUCCAACCGACCCUGA miR-24-1 CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCA 53UUUUACACACUGGCUCAGUUCAGCAGGAACAGG AG miR-24-2CUCUGCCUCCCGUGCCUACUGAGCUGAAACACAG 54 UUGGUUUGUGUACACUGGCUCAGUUCAGCAGGAACAGGG miR-24-19 CCCUGGGCUCUGCCUCCCGUGCCUACUGAGCUGA 55AACACAGUUGGUUUGUGUACACUGGCUCAGUUC AGCAGGAACAGGGG miR-24-9CCCUCCGGUGCCUACUGAGCUGAUAUCAGUUCU 56 CAUUUUACACACUGGCUCAGUUCAGCAGGAACAGCAUC miR-25 GGCCAGUGUUGAGAGGCGGAGACUUGGGCAAUU 57GCUGGACGCUGCCCUGGGCAUUGCACUUGUCUC GGUCUGACAGUGCCGGCC miR-26aAGGCCGUGGCCUCGUUCAAGUAAUCCAGGAUAG 58 GCUGUGCAGGUCCCAAUGGCCUAUCUUGGUUACUUGCACGGGGACGCGGGCCU miR-26a-1 GUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGU 59GCAGGUCCCAAUGGGCCUAUUCUUGGUUACUUG CACGGGGACGC miR-26a-2GGCUGUGGCUGGAUUCAAGUAAUCCAGGAUAGG 60 CUGUUUCCAUCUGUGAGGCCUAUUCUUGAUUACUUGUUUCUGGAGGCAGCU miR-26b CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUU 61GUGUGCUGUCCAGCCUGUUCUCCAUUACUUGGC UCGGGGACCGG miR-27aCUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAGG 62 GUCCACACCAAGUCGUGUUCACAGUGGCUAAGUUCCGCCCCCCAG miR-27b-1 AGGUGCAGAGCUUAGCUGAUUGGUGAACAGUGA 63UUGGUUUCCGCUUUGUUCACAGUGGCUAAGUUC UGCACCU miR-27b-2ACCUCUCUAACAAGGUGCAGAGCUUAGCUGAUU 64 GGUGAACAGUGAUUGGUUUCCGCUUUGUUCACAGUGGCUAAGUUCUGCACCUGAAGAGAAGGUG miR-27-19CCUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAG 65 GGUCCACACCAAGUCGUGUUCACAGUGGCUAAGUUCCGCCCCCCAGG miR-28 GGUCCUUGCCCUCAAGGAGCUCACAGUCUAUUG 66AGUUACCUUUCUGACUUUCCCACUAGAUUGUGA GCUCCUGGAGGGCAGGCACU miR-29a-2CCUUCUGUGACCCCUUAGAGGAUGACUGAUUUC 67 UUUUGGUGUUCAGAGUCAAUAUAAUUUUCUAGCACCAUCUGAAAUCGGUUAUAAUGAUUGGGGAAG AGCACCAUG miR-29aAUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUA 68 UAAUUUUCUAGCACCAUCUGAAAUCGGUUAUmiR-29b-1 CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGA 69UUUAAAUAGUGAUUGUCUAGCACCAUUUGAAAU CAGUGUUCUUGGGGG miR-29b-2CUUCUGGAAGCUGGUUUCACAUGGUGGCUUAGA 70 UUUUUCCAUCUUUGUAUCUAGCACCAUUUGAAAUCAGUGUUUUAGGAG miR-29c ACCACUGGCCCAUCUCUUACACAGGCUGACCGAU 71UUCUCCUGGUGUUCAGAGUCUGUUUUUGUCUAG CACCAUUUGAAAUCGGUUAUGAUGUAGGGGGAAAAGCAGCAGC miR-30a GCGACUGUAAACAUCCUCGACUGGAAGCUGUGA 72AGCCACAGAUGGGCUUUCAGUCGGAUGUUUGCA GCUGC miR-30b-1AUGUAAACAUCCUACACUCAGCUGUAAUACAUG 73 GAUUGGCUGGGAGGUGGAUGUUUACGUmiR-30b-2 ACCAAGUUUCAGUUCAUGUAAACAUCCUACACU 74CAGCUGUAAUACAUGGAUUGGCUGGGAGGUGGA UGUUUACUUCAGCUGACUUGGA miR-30cAGAUACUGUAAACAUCCUACACUCUCAGCUGUG 75 GAAAGUAAGAAAGCUGGGAGAAGGCUGUUUACUCUUUCU miR-30d GUUGUUGUAAACAUCCCCGACUGGAAGCUGUAA 76GACACAGCUAAGCUUUCAGUCAGAUGUUUGCUG CUAC miR-30eCUGUAAACAUCCUUGACUGGAAGCUGUAAGGUG 77 UUCAGAGGAGCUUUCAGUCGGAUGUUUACAGmiR-31 GGAGAGGAGGCAAGAUGCUGGCAUAGCUGUUGA 78ACUGGGAACCUGCUAUGCCAACAUAUUGCCAUC UUUCC miR-32GGAGAUAUUGCACAUUACUAAGUUGCAUGUUGU 79 CACGGCCUCAAUGCAAUUUAGUGUGUGUGAUAUUUUC miR-33b GGGGGCCGAGAGAGGCGGGCGGCCCCGCGGUGC 80AUUGCUGUUGCAUUGCACGUGUGUGAGGCGGGU GCAGUGCCUCGGCAGUGCAGCCCGGAGCCGGCCCCUGGCACCAC miR-33b-2 ACCAAGUUUCAGUUCAUGUAAACAUCCUACACU 81CAGCUGUAAUACAUGGAUUGGCUGGGAGGUGGA UGUUUACUUCAGCUGACUUGGA miR-33CUGUGGUGCAUUGUAGUUGCAUUGCAUGUUCUG 82 GUGGUACCCAUGCAAUGUUUCCACAGUGCAUCACAG miR-34-a GGCCAGCUGUGAGUGUUUCUUUGGCAGUGUCUU 83AGCUGGUUGUUGUGAGCAAUAGUAAGGAAGCAA UCAGCAAGUAUACUGCCCUAGAAGUGCUGCACGUUGUGGGGCCC miR-34-b GUGCUCGGUUUGUAGGCAGUGUCAUUAGCUGAU 84UGUACUGUGGUGGUUACAAUCACUAACUCCACU GCCAUCAAAACAAGGCAC miR-34-cAGUCUAGUUACUAGGCAGUGUAGUUAGCUGAUU 85 GCUAAUAGUACCAAUCACUAACCACACGGCCAGGUAAAAAGAUU miR-91-13 UCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGU 86AGUGAUAUGUGCAUCUACUGCAGUGAAGGCACU UGUAGCAUUAUGGUGA miR-92-1CUUUCUACACAGGUUGGGAUCGGUUGCAAUGCU 87 GUGUUUCUGUAUGGUAUUGCACUUGUCCCGGCCUGUUGAGUUUGG miR-92-2 UCAUCCCUGGGUGGGGAUUUGUUGCAUUACUUG 88UGUUCUAUAUAAAGUAUUGCACUUGUCCCGGCC UGUGGAAGA miR-93-1CUGGGGGCUCCAAAGUGCUGUUCGUGCAGGUAG 89 (miR-93-2)UGUGAUUACCCAACCUACUGCUGAGCUAGCACU UCCCGAGCCCCCGG miR-95-4AACACAGUGGGCACUCAAUAAAUGUCUGUUGAA 90 UUGAAAUGCGUUACAUUCAACGGGUAUUUAUUGAGCACCCACUCUGUG miR-96-7 UGGCCGAUUUUGGCACUAGCACAUUUUUGCUUG 91UGUCUCUCCGCUCUGAGCAAUCAUGUGCAGUGC CAAUAUGGGAAA miR-97-6GUGAGCGACUGUAAACAUCCUCGACUGGAAGCU 92 (miR-30*)GUGAAGCCACAGAUGGGCUUUCAGUCGGAUGUU UGCAGCUGCCUACU miR-98GUGAGGUAGUAAGUUGUAUUGUUGUGGGGUAGG 93 GAUAUUAGGCCCCAAUUAGAAGAUAACUAUACAACUUACUACUUUCC miR-99b GGCACCCACCCGUAGAACCGACCUUGCGGGGCCU 94UCGCCGCACACAAGCUCGUGUCUGUGGGUCCGU GUC miR-99aCCCAUUGGCAUAAACCCGUAGAUCCGAUCUUGU 95 GGUGAAGUGGACCGCACAAGCUCGCUUCUAUGGGUCUGUGUCAGUGUG miR-100-1/2 AAGAGAGAAGAUAUUGAGGCCUGUUGCCACAAA 96CCCGUAGAUCCGAACUUGUGGUAUUAGUCCGCA CAAGCUUGUAUCUAUAGGUAUGUGUCUGUUAGGCAAUCUCAC miR-100-11 CCUGUUGCCACAAACCCGUAGAUCCGAACUUGU 97GGUAUUAGUCCGCACAAGCUUGUAUCUAUAGGU AUGUGUCUGUUAGG miR-101-1/2AGGCUGCCCUGGCUCAGUUAUCACAGUGCUGAU 98 GCUGUCUAUUCUAAAGGUACAGUACUGUGAUAACUGAAGGAUGGCAGCCAUCUUACCUUCCAUCAG AGGAGCCUCAC miR-101UCAGUUAUCACAGUGCUGAUGCUGUCCAUUCUA 99 AAGGUACAGUACUGUGAUAACUGA miR-101-1UGCCCUGGCUCAGUUAUCACAGUGCUGAUGCUG 100 UCUAUUCUAAAGGUACAGUACUGUGAUAACUGAAGGAUGGCA miR-101-2 ACUGUCCUUUUUCGGUUAUCAUGGUACCGAUGC 101UGUAUAUCUGAAAGGUACAGUACUGUGAUAACU GAAGAAUGGUGGU miR-101-9UGUCCUUUUUCGGUUAUCAUGGUACCGAUGCUG 102 UAUAUCUGAAAGGUACAGUACUGUGAUAACUGAAGAAUGGUG miR-102-1 CUUCUGGAAGCUGGUUUCACAUGGUGGCUUAGA 103UUUUUCCAUCUUUGUAUCUAGCACCAUUUGAAA UCAGUGUUUUAGGAG miR-102-7.1CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGA 104 (miR-102-UUUAAAUAGUGAUUGUCUAGCACCAUUUGAAAU 7.2) CAGUGUUCUUGGGGG miR-103-2UUGUGCUUUCAGCUUCUUUACAGUGCUGCCUUG 105 UAGCAUUCAGGUCAAGCAACAUUGUACAGGGCUAUGAAAGAACCA miR-103-1 UACUGCCCUCGGCUUCUUUACAGUGCUGCCUUG 106UUGCAUAUGGAUCAAGCAGCAUUGUACAGGGCU AUGAAGGCAUUG miR-104-17AAAUGUCAGACAGCCCAUCGACUGGUGUUGCCA 107 UGAGAUUCAACAGUCAACAUCAGUCUGAUAAGCUACCCGACAAGG miR-105-1 UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUG 108GUGGCUGCUCAUGCACCACGGAUGUUUGAGCAU GUGCUACGGUGUCUA miR-105-2UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUG 109 GUGGCUGCUUAUGCACCACGGAUGUUUGAGCAUGUGCUAUGGUGUCUA miR-106-a CCUUGGCCAUGUAAAAGUGCUUACAGUGCAGGU 110AGCUUUUUGAGAUCUACUGCAAUGUAAGCACUU CUUACAUUACCAUGG miR-106-bCCUGCCGGGGCUAAAGUGCUGACAGUGCAGAUA 111 GUGGUCCUCUCCGUGCUACCGCACUGUGGGUACUUGCUGCUCCAGCAGG miR-107 CUCUCUGCUUUCAGCUUCUUUACAGUGUUGCCU 112UGUGGCAUGGAGUUCAAGCAGCAUUGUACAGGG CUAUCAAAGCACAGA miR-108-1-ACACUGCAAGAACAAUAAGGAUUUUUAGGGGCA 113 smallUUAUGACUGAGUCAGAAAACACAGCUGCCCCUG AAAGUCCCUCAUUUUUCUUGCUGU miR-108-2-ACUGCAAGAGCAAUAAGGAUUUUUAGGGGCAUU 114 smallAUGAUAGUGGAAUGGAAACACAUCUGCCCCCAA AAGUCCCUCAUUUU miR-122a-1CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGU 115 UUGUGUCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCUACUGCUAGGC miR-122a-2 AGCUGUGGAGUGUGACAAUGGUGUUUGUGUCCA 116AACUAUCAAACGCCAUUAUCACACUAAAUAGCU miR-123ACAUUAUUACUUUUGGUACGCGCUGUGACACUU 117 CAAACUCGUACCGUGAGUAAUAAUGCGCmiR-124a-1 AGGCCUCUCUCUCCGUGUUCACAGCGGACCUUG 118AUUUAAAUGUCCAUACAAUUAAGGCACGCGGUG AAUGCCAAGAAUGGGGCUG miR-124a-2AUCAAGAUUAGAGGCUCUGCUCUCCGUGUUCAC 119 AGCGGACCUUGAUUUAAUGUCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGCGGAGCCUACGGC UGCACUUGAAG miR-124a-3UGAGGGCCCCUCUGCGUGUUCACAGCGGACCUU 120 GAUUUAAUGUCUAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGAGGCGCCUCC miR-124a CUCUGCGUGUUCACAGCGGACCUUGAUUUAAUG 121UCUAUACAAUUAAGGCACGCGGUGAAUGCCAAG AG miR-124bCUCUCCGUGUUCACAGCGGACCUUGAUUUAAUG 122 UCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGmiR-125a-1 UGCCAGUCUCUAGGUCCCUGAGACCCUUUAACC 123UGUGAGGACAUCCAGGGUCACAGGUGAGGUUCU UGGGAGCCUGGCGUCUGGCC miR-125a-2GGUCCCUGAGACCCUUUAACCUGUGAGGACAUC 124 CAGGGUCACAGGUGAGGUUCUUGGGAGCCUGGmiR-125b-1 UGCGCUCCUCUCAGUCCCUGAGACCCUAACUUGU 125GAUGUUUACCGUUUAAAUCCACGGGUUAGGCUC UUGGGAGCUGCGAGUCGUGCU miR-125b-2ACCAGACUUUUCCUAGUCCCUGAGACCCUAACU 126 UGUGAGGUAUUUUAGUAACAUCACAAGUCAGGCUCUUGGGACCUAGGCGGAGGGGA miR-126-1 CGCUGGCGACGGGACAUUAUUACUUUUGGUACG 127CGCUGUGACACUUCAAACUCGUACCGUGAGUAA UAAUGCGCCGUCCACGGCA miR-126-2ACAUUAUUACUUUUGGUACGCGCUGUGACACUU 128 CAAACUCGUACCGUGAGUAAUAAUGCGCmiR-127-1 UGUGAUCACUGUCUCCAGCCUGCUGAAGCUCAG 129AGGGCUCUGAUUCAGAAAGAUCAUCGGAUCCGU CUGAGCUUGGCUGGUCGGAAGUCUCAUCAUCmiR-127-2 CCAGCCUGCUGAAGCUCAGAGGGCUCUGAUUCA 130GAAAGAUCAUCGGAUCCGUCUGAGCUUGGCUGG UCGG miR-128aUGAGCUGUUGGAUUCGGGGCCGUAGCACUGUCU 131 GAGAGGUUUACAUUUCUCACAGUGAACCGGUCUCUUUUUCAGCUGCUUC miR-128b GCCCGGCAGCCACUGUGCAGUGGGAAGGGGGGC 132CGAUACACUGUACGAGAGUGAGUAGCAGGUCUC ACAGUGAACCGGUCUCUUUCCCUACUGUGUCACACUCCUAAUGG miR-128 GUUGGAUUCGGGGCCGUAGCACUGUCUGAGAGG 133UUUACAUUUCUCACAGUGAACCGGUCUCUUUUU CAGC miR-129-1UGGAUCUUUUUGCGGUCUGGGCUUGCUGUUCCU 134 CUCAACAGUAGUCAGGAAGCCCUUACCCCAAAAAGUAUCUA miR-129-2 UGCCCUUCGCGAAUCUUUUUGCGGUCUGGGCUU 135GCUGUACAUAACUCAAUAGCCGGAAGCCCUUAC CCCAAAAAGCAUUUGCGGAGGGCG miR-130aUGCUGCUGGCCAGAGCUCUUUUCACAUUGUGCU 136 ACUGUCUGCACCUGUCACUAGCAGUGCAAUGUUAAAAGGGCAUUGGCCGUGUAGUG miR-131-1 GCCAGGAGGCGGGGUUGGUUGUUAUCUUUGGUU 137AUCUAGCUGUAUGAGUGGUGUGGAGUCUUCAUA AAGCUAGAUAACCGAAAGUAAAAAUAACCCCAUACACUGCGCAG miR-131-3 CACGGCGCGGCAGCGGCACUGGCUAAGGGAGGC 138CCGUUUCUCUCUUUGGUUAUCUAGCUGUAUGAG UGCCACAGAGCCGUCAUAAAGCUAGAUAACCGAAAGUAGAAAUG miR-131 GUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUG 139UAUUGGUCUUCAUAAAGCUAGAUAACCGAAAGU AAAAAC miR-132-1CCGCCCCCGCGUCUCCAGGGCAACCGUGGCUUUC 140 GAUUGUUACUGUGGGAACUGGAGGUAACAGUCUACAGCCAUGGUCGCCCCGCAGCACGCCCACGCGC miR-132-2GGGCAACCGUGGCUUUCGAUUGUUACUGUGGGA 141 ACUGGAGGUAACAGUCUACAGCCAUGGUCGCCCmiR-133a-1 ACAAUGCUUUGCUAGAGCUGGUAAAAUGGAACC 142AAAUCGCCUCUUCAAUGGAUUUGGUCCCCUUCA ACCAGCUGUAGCUAUGCAUUGA miR-133a-2GGGAGCCAAAUGCUUUGCUAGAGCUGGUAAAAU 143 GGAACCAAAUCGACUGUCCAAUGGAUUUGGUCCCCUUCAACCAGCUGUAGCUGUGCAUUGAUGGCG CCG miR-133GCUAGAGCUGGUAAAAUGGAACCAAAUCGCCUC 144 UUCAAUGGAUUUGGUCCCCUUCAACCAGCUGUAGC miR-133b CCUCAGAAGAAAGAUGCCCCCUGCUCUGGCUGG 145UCAAACGGAACCAAGUCCGUCUUCCUGAGAGGU UUGGUCCCCUUCAACCAGCUACAGCAGGGCUGGCAAUGCCCAGUCCUUGGAGA miR-133b- GCCCCCUGCUCUGGCUGGUCAAACGGAACCAAG 146small UCCGUCUUCCUGAGAGGUUUGGUCCCCUUCAAC CAGCUACAGCAGGG miR-134-1CAGGGUGUGUGACUGGUUGACCAGAGGGGCAUG 147 CACUGUGUUCACCCUGUGGGCCACCUAGUCACCAACCCUC miR-134-2 AGGGUGUGUGACUGGUUGACCAGAGGGGCAUGC 148ACUGUGUUCACCCUGUGGGCCACCUAGUCACCA ACCCU miR-135a-1AGGCCUCGCUGUUCUCUAUGGCUUUUUAUUCCU 149 AUGUGAUUCUACUGCUCACUCAUAUAGGGAUUGGAGCCGUGGCGCACGGCGGGGACA miR-135a-2 AGAUAAAUUCACUCUAGUGCUUUAUGGCUUUUU150 (miR-135-2) AUUCCUAUGUGAUAGUAAUAAAGUCUCAUGUAGGGAUGGAAGCCAUGAAAUACAUUGUGAAAAAUCA miR-135CUAUGGCUUUUUAUUCCUAUGUGAUUCUACUGC 151 UCACUCAUAUAGGGAUUGGAGCCGUGGmiR-135b CACUCUGCUGUGGCCUAUGGCUUUUCAUUCCUA 152UGUGAUUGCUGUCCCAAACUCAUGUAGGGCUAA AAGCCAUGGGCUACAGUGAGGGGCGAGCUCCmiR-136-1 UGAGCCCUCGGAGGACUCCAUUUGUUUUGAUGA 153UGGAUUCUUAUGCUCCAUCAUCGUCUCAAAUGA GUCUUCAGAGGGUUCU miR-136-2GAGGACUCCAUUUGUUUUGAUGAUGGAUUCUUA 154 UGCUCCAUCAUCGUCUCAAAUGAGUCUUCmiR-137 CUUCGGUGACGGGUAUUCUUGGGUGGAUAAUAC 155GGAUUACGUUGUUAUUGCUUAAGAAUACGCGUA GUCGAGG miR-138-1CCCUGGCAUGGUGUGGUGGGGCAGCUGGUGUUG 156 UGAAUCAGGCCGUUGCCAAUCAGAGAACGGCUACUUCACAACACCAGGGCCACACCACACUACAGG miR-138-2CGUUGCUGCAGCUGGUGUUGUGAAUCAGGCCGA 157 CGAGCAGCGCAUCCUCUUACCCGGCUAUUUCACGACACCAGGGUUGCAUCA miR-138 CAGCUGGUGUUGUGAAUCAGGCCGACGAGCAGC 158GCAUCCUCUUACCCGGCUAUUUCACGACACCAGG GUUG miR-139GUGUAUUCUACAGUGCACGUGUCUCCAGUGUGG 159 CUCGGAGGCUGGAGACGCGGCCCUGUUGGAGUAAC miR-140 UGUGUCUCUCUCUGUGUCCUGCCAGUGGUUUUA 160CCCUAUGGUAGGUUACGUCAUGCUGUUCUACCA CAGGGUAGAACCACGGACAGGAUACCGGGGCACCmiR-140as UCCUGCCAGUGGUUUUACCCUAUGGUAGGUUAC 161GUCAUGCUGUUCUACCACAGGGUAGAACCACGG ACAGGA miR-140sCCUGCCAGUGGUUUUACCCUAUGGUAGGUUACG 162 UCAUGCUGUUCUACCACAGGGUAGAACCACGGACAGG miR-141-1 CGGCCGGCCCUGGGUCCAUCUUCCAGUACAGUG 163UUGGAUGGUCUAAUUGUGAAGCUCCUAACACUG UCUGGUAAAGAUGGCUCCCGGGUGGGUUCmiR-141-2 GGGUCCAUCUUCCAGUACAGUGUUGGAUGGUCU 164AAUUGUGAAGCUCCUAACACUGUCUGGUAAAGA UGGCCC miR-142ACCCAUAAAGUAGAAAGCACUACUAACAGCACU 165 GGAGGGUGUAGUGUUUCCUACUUUAUGGAUGmiR-143-1 GCGCAGCGCCCUGUCUCCCAGCCUGAGGUGCAGU 166GCUGCAUCUCUGGUCAGUUGGGAGUCUGAGAUG AAGCACUGUAGCUCAGGAAGAGAGAAGUUGUUCUGCAGC miR-143-2 CCUGAGGUGCAGUGCUGCAUCUCUGGUCAGUUG 167GGAGUCUGAGAUGAAGCACUGUAGCUCAGG miR-144-1UGGGGCCCUGGCUGGGAUAUCAUCAUAUACUGU 168 AAGUUUGCGAUGAGACACUACAGUAUAGAUGAUGUACUAGUCCGGGCACCCCC miR-144-2 GGCUGGGAUAUCAUCAUAUACUGUAAGUUUGCG 169AUGAGACACUACAGUAUAGAUGAUGUACUAGUC miR-145-1CACCUUGUCCUCACGGUCCAGUUUUCCCAGGAA 170 UCCCUUAGAUGCUAAGAUGGGGAUUCCUGGAAAUACUGUUCUUGAGGUCAUGGUU miR-145-2 CUCACGGUCCAGUUUUCCCAGGAAUCCCUUAGA 171UGCUAAGAUGGGGAUUCCUGGAAAUACUGUUCU UGAG miR-146-1CCGAUGUGUAUCCUCAGCUUUGAGAACUGAAUU 172 CCAUGGGUUGUGUCAGUGUCAGACCUCUGAAAUUCAGUUCUUCAGCUGGGAUAUCUCUGUCAUCGU miR-146-2AGCUUUGAGAACUGAAUUCCAUGGGUUGUGUCA 173 GUGUCAGACCUGUGAAAUUCAGUUCUUCAGCUmiR-147 AAUCUAAAGACAACAUUUCUGCACACACACCAG 174ACUAUGGAAGCCAGUGUGUGGAAAUGCUUCUGC UAGAUU miR-148aGAGGCAAAGUUCUGAGACACUCCGACUCUGAGU 175 (miR-148)AUGAUAGAAGUCAGUGCACUACAGAACUUUGUC UC miR-148bCAAGCACGAUUAGCAUUUGAGGUGAAGUUCUGU 176 UAUACACUCAGGCUGUGGCUCUCUGAAAGUCAGUGCAUCACAGAACUUUGUCUCGAAAGCUUUCUA miR-148b-AAGCACGAUUAGCAUUUGAGGUGAAGUUCUGUU 177 smallAUACACUCAGGCUGUGGCUCUCUGAAAGUCAGU GCAU miR-149-1GCCGGCGCCCGAGCUCUGGCUCCGUGUCUUCACU 178 CCCGUGCUUGUCCGAGGAGGGAGGGAGGGACGGGGGCUGUGCUGGGGCAGCUGGA miR-149-2 GCUCUGGCUCCGUGUCUUCACUCCCGUGCUUGUC 179CGAGGAGGGAGGGAGGGAC miR-150-1 CUCCCCAUGGCCCUGUCUCCCAACCCUUGUACCA 180GUGCUGGGCUCAGACCCUGGUACAGGCCUGGGG GACAGGGACCUGGGGAC miR-150-2CCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCU 181 CAGACCCUGGUACAGGCCUGGGGGACAGGGmiR-151 UUUCCUGCCCUCGAGGAGCUCACAGUCUAGUAU 182GUCUCAUCCCCUACUAGACUGAAGCUCCUUGAG GACAGG miR-151-2CCUGUCCUCAAGGAGCUUCAGUCUAGUAGGGGA 183 UGAGACAUACUAGACUGUGAGCUCCUCGAGGGCAGG miR-152-1 UGUCCCCCCCGGCCCAGGUUCUGUGAUACACUCC 184GACUCGGGCUCUGGAGCAGUCAGUGCAUGACAG AACUUGGGCCCGGAAGGACC miR-152-2GGCCCAGGUUCUGUGAUACACUCCGACUCGGGC 185 UCUGGAGCAGUCAGUGCAUGACAGAACUUGGGCCCCGG miR-153-1-1 CUCACAGCUGCCAGUGUCAUUUUUGUGAUCUGC 186AGCUAGUAUUCUCACUCCAGUUGCAUAGUCACA AAAGUGAUCAUUGGCAGGUGUGGC miR-153-1-2UCUCUCUCUCCCUCACAGCUGCCAGUGUCAUUGU 187 CACAAAAGUGAUCAUUGGCAGGUGUGGCUGCUGCAUG miR-153-2-1 AGCGGUGGCCAGUGUCAUUUUUGUGAUGUUGCA 188GCUAGUAAUAUGAGCCCAGUUGCAUAGUCACAA AAGUGAUCAUUGGAAACUGUG miR-153-2-2CAGUGUCAUUUUUGUGAUGUUGCAGCUAGUAAU 189 AUGAGCCCAGUUGCAUAGUCACAAAAGUGAUCAUUG miR-154-1 GUGGUACUUGAAGAUAGGUUAUCCGUGUUGCCU 190UCGCUUUAUUUGUGACGAAUCAUACACGGUUGA CCUAUUUUUCAGUACCAA miR-154-2GAAGAUAGGUUAUCCGUGUUGCCUUCGCUUUAU 191 UUGUGACGAAUCAUACACGGUUGACCUAUUUUUmiR-155 CUGUUAAUGCUAAUCGUGAUAGGGGUUUUUGCC 192UCCAACUGACUCCUACAUAUUAGCAUUAACAG miR-156 =CCUAACACUGUCUGGUAAAGAUGGCUCCCGGGU 193 miR-GGGUUCUCUCGGCAGUAACCUUCAGGGAGCCCU 157 = overlap GAAGACCAUGGAGGAC miR-141miR-158- GCCGAGACCGAGUGCACAGGGCUCUGACCUAUG 194 small = miR-AAUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGC 192 UGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCUCAAUGCCAGC miR-159-1- UCCCGCCCCCUGUAACAGCAACUCCAUGUGGAAG 195 smallUGCCCACUGGUUCCAGUGGGGCUGCUGUUAUCU GGGGCGAGGGCCA miR-161-AAAGCUGGGUUGAGAGGGCGAAAAAGGAUGAGG 196 smallUGACUGGUCUGGGCUACGCUAUGCUGCGGCGCU CGGG miR-163-1b-CAUUGGCCUCCUAAGCCAGGGAUUGUGGGUUCG 197 smallAGUCCCACCCGGGGUAAAGAAAGGCCGAAUU miR-163-3-CCUAAGCCAGGGAUUGUGGGUUCGAGUCCCACC 198 smallUGGGGUAGAGGUGAAAGUUCCUUUUACGGAAUU UUUU miR-162CAAUGUCAGCAGUGCCUUAGCAGCACGUAAAUA 199 UUGGCGUUAAGAUUCUAAAAUUAUCUCCAGUAUUAACUGUGCUGCUGAAGUAAGGUUGACCAUACU CUACAGUUG miR-175-GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUA 200 small = miR-GAUGAUUGUGCAUUGUUUCAAAAUGGUGCCCUA 224 GUGACUACAAAGCCC miR-177-ACGCAAGUGUCCUAAGGUGAGCUCAGGGAGCAC 201 smallAGAAACCUCCAGUGGAACAGAAGGGCAAAAGCU CAUU miR-180-CAUGUGUCACUUUCAGGUGGAGUUUCAAGAGUC 202 smallCCUUCCUGGUUCACCGUCUCCUUUGCUCUUCCAC AAC miR-181aAGAAGGGCUAUCAGGCCAGCCUUCAGAGGACUC 203 CAAGGAACAUUCAACGCUGUCGGUGAGUUUGGGAUUUGAAAAAACCACUGACCGUUGACUGUACCU UGGGGUCCUUA miR-181b-1CCUGUGCAGAGAUUAUUUUUUAAAAGGUCACAA 204 UCAACAUUCAUUGCUGUCGGUGGGUUGAACUGUGUGGACAAGCUCACUGAACAAUGAAUGCAACUG UGGCCCCGCUU miR-181b-2CUGAUGGCUGCACUCAACAUUCAUUGCUGUCGG 205 UGGGUUUGAGUCUGAAUCAACUCACUGAUCAAUGAAUGCAAACUGCGGACCAAACA miR-181c CGGAAAAUUUGCCAAGGGUUUGGGGGAACAUUC 206AACCUGUCGGUGAGUUUGGGCAGCUCAGGCAAA CCAUCGACCGUUGAGUGGACCCUGAGGCCUGGAAUUGCCAUCCU miR-182-as GAGCUGCUUGCCUCCCCCCGUUUUUGGCAAUGG 207UAGAACUCACACUGGUGAGGUAACAGGAUCCGG UGGUUCUAGACUUGCCAACUAUGGGGCGAGGACUCAGCCGGCAC miR-182 UUUUUGGCAAUGGUAGAACUCACACUGGUGAGG 208UAACAGGAUCCGGUGGUUCUAGACUUGCCAACU AUGG miR-183CCGCAGAGUGUGACUCCUGUUCUGUGUAUGGCA 209 CUGGUAGAAUUCACUGUGAACAGUCUCAGUCAGUGAAUUACCGAAGGGCCAUAAACAGAGCAGAGA CAGAUCCACGA miR-184-1CCAGUCACGUCCCCUUAUCACUUUUCCAGCCCAG 210 CUUUGUGACUGUAAGUGUUGGACGGAGAACUGAUAAGGGUAGGUGAUUGA miR-184-2 CCUUAUCACUUUUCCAGCCCAGCUUUGUGACUG 211UAAGUGUUGGACGGAGAACUGAUAAGGGUAGG miR-185-1AGGGGGCGAGGGAUUGGAGAGAAAGGCAGUUCC 212 UGAUGGUCCCCUCCCCAGGGGCUGGCUUUCCUCUGGUCCUUCCCUCCCA miR-185-2 AGGGAUUGGAGAGAAAGGCAGUUCCUGAUGGUC 213CCCUCCCCAGGGGCUGGCUUUCCUCUGGUCCUU miR-186-1UGCUUGUAACUUUCCAAAGAAUUCUCCUUUUGG 214 GCUUUCUGGUUUUAUUUUAAGCCCAAAGGUGAAUUUUUUGGGAAGUUUGAGCU miR-186-2 ACUUUCCAAAGAAUUCUCCUUUUGGGCUUUCUG 215GUUUUAUUUUAAGCCCAAAGGUGAAUUUUUUGG GAAGU miR-187GGUCGGGCUCACCAUGACACAGUGUGAGACUCG 216 GGCUACAACACAGGACCCGGGGCGCUGCUCUGACCCCUCGUGUCUUGUGUUGCAGCCGGAGGGACG CAGGUCCGCA miR-188-1UGCUCCCUCUCUCACAUCCCUUGCAUGGUGGAG 217 GGUGAGCUUUCUGAAAACCCCUCCCACAUGCAGGGUUUGCAGGAUGGCGAGCC miR-188-2 UCUCACAUCCCUUGCAUGGUGGAGGGUGAGCUU 218UCUGAAAACCCCUCCCACAUGCAGGGUUUGCAG GA miR-189-1CUGUCGAUUGGACCCGCCCUCCGGUGCCUACUGA 219 GCUGAUAUCAGUUCUCAUUUUACACACUGGCUCAGUUCAGCAGGAACAGGAGUCGAGCCCUUGAGC AA miR-189-2CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCA 220 UUUUACACACUGGCUCAGUUCAGCAGGAACAGGAG miR-190-1 UGCAGGCCUCUGUGUGAUAUGUUUGAUAUAUUA 221GGUUGUUAUUUAAUCCAACUAUAUAUCAAACAU AUUCCUACAGUGUCUUGCC miR-190-2CUGUGUGAUAUGUUUGAUAUAUUAGGUUGUUAU 222 UUAAUCCAACUAUAUAUCAAACAUAUUCCUACAGmiR-191-1 CGGCUGGACAGCGGGCAACGGAAUCCCAAAAGC 223AGCUGUUGUCUCCAGAGCAUUCCAGCUGCGCUU GGAUUUCGUCCCCUGCUCUCCUGCCU miR-191-2AGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGU 224 CUCCAGAGCAUUCCAGCUGCGCUUGGAUUUCGUCCCCUGCU miR-192-2/3 CCGAGACCGAGUGCACAGGGCUCUGACCUAUGA 225AUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGCU GCCAAUUCCAUAGGUCACAGGUAUGUUCGCCUCAAUGCCAG miR-192 GCCGAGACCGAGUGCACAGGGCUCUGACCUAUG 226AAUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGC UGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCUCAAUGCCAGC miR-193-1 CGAGGAUGGGAGCUGAGGGCUGGGUCUUUGCGG 227GCGAGAUGAGGGUGUCGGAUCAACUGGCCUACA AAGUCCCAGUUCUCGGCCCCCG miR-193-2GCUGGGUCUUUGCGGGCGAGAUGAGGGUGUCGG 228 AUCAACUGGCCUACAAAGUCCCAGUmiR-194-1 AUGGUGUUAUCAAGUGUAACAGCAACUCCAUGU 229GGACUGUGUACCAAUUUCCAGUGGAGAUGCUGU UACUUUUGAUGGUUACCAA miR-194-2GUGUAACAGCAACUCCAUGUGGACUGUGUACCA 230 AUUUCCAGUGGAGAUGCUGUUACUUUUGAUmiR-195-1 AGCUUCCCUGGCUCUAGCAGCACAGAAAUAUUG 231GCACAGGGAAGCGAGUCUGCCAAUAUUGGCUGU GCUGCUCCAGGCAGGGUGGUG miR-195-2UAGCAGCACAGAAAUAUUGGCACAGGGAAGCGA 232 GUCUGCCAAUAUUGGCUGUGCUGCUmiR-196-1 CUAGAGCUUGAAUUGGAACUGCUGAGUGAAUUA 233GGUAGUUUCAUGUUGUUGGGCCUGGGUUUCUGA ACACAACAACAUUAAACCACCCGAUUCACGGCAGUUACUGCUCC miR-196a-1 GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGG 234GUUUCUGAACACAACAACAUUAAACCACCCGAU UCAC miR-196a-2UGCUCGCUCAGCUGAUCUGUGGCUUAGGUAGUU 235 (miR-196-2)UCAUGUUGUUGGGAUUGAGUUUUGAACUCGGCA ACAAGAAACUGCCUGAGUUACAUCAGUCGGUUUUCGUCGAGGGC miR-196 GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGG 236GUUUCUGAACACAACAACAUUAAACCACCCGAU UCAC miR-196bACUGGUCGGUGAUUUAGGUAGUUUCCUGUUGUU 237 GGGAUCCACCUUUCUCUCGACAGCACGACACUGCCUUCAUUACUUCAGUUG miR-197 GGCUGUGCCGGGUAGAGAGGGCAGUGGGAGGUA 238AGAGCUCUUCACCCUUCACCACCUUCUCCACCCA GCAUGGCC miR-197-2GUGCAUGUGUAUGUAUGUGUGCAUGUGCAUGUG 239 UAUGUGUAUGAGUGCAUGCGUGUGUGCmiR-198 UCAUUGGUCCAGAGGGGAGAUAGGUUCCUGUGA 240UUUUUCCUUCUUCUCUAUAGAAUAAAUGA miR-199a-1GCCAACCCAGUGUUCAGACUACCUGUUCAGGAG 241 GCUCUCAAUGUGUACAGUAGUCUGCACAUUGGUUAGGC miR-199a-2 AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCC 242AGUGUUCAGACUACCUGUUCAGGACAAUGCCGU UGUACAGUAGUCUGCACAUUGGUUAGACUGGGCAAGGGAGAGCA miR-199b CCAGAGGACACCUCCACUCCGUCUACCCAGUGUU 243UAGACUAUCUGUUCAGGACUCCCAAAUUGUACA GUAGUCUGCACAUUGGUUAGGCUGGGCUGGGUUAGACCCUCGG miR-199s GCCAACCCAGUGUUCAGACUACCUGUUCAGGAG 244GCUCUCAAUGUGUACAGUAGUCUGCACAUUGGU UAGGC miR-200aGCCGUGGCCAUCUUACUGGGCAGCAUUGGAUGG 245 AGUCAGGUCUCUAAUACUGCCUGGUAAUGAUGACGGC miR-200b CCAGCUCGGGCAGCCGUGGCCAUCUUACUGGGC 246AGCAUUGGAUGGAGUCAGGUCUCUAAUACUGCC UGGUAAUGAUGACGGCGGAGCCCUGCACG miR-200cCCCUCGUCUUACCCAGCAGUGUUUGGGUGCGGU 247 UGGGAGUCUCUAAUACUGCCGGGUAAUGAUGGAGG miR-202 GUUCCUUUUUCCUAUGCAUAUACUUCUUUGAGG 248AUCUGGCCUAAAGAGGUAUAGGGCAUGGGAAGA UGGAGC miR-203GUGUUGGGGACUCGCGCGCUGGGUCCAGUGGUU 249 CUUAACAGUUCAACAGUUCUGUAGCGCAAUUGUGAAAUGUUUAGGACCACUAGACCCGGCGGGCGC GGCGACAGCGA miR-204GGCUACAGUCUUUCUUCAUGUGACUCGUGGACU 250 UCCCUUUGUCAUCCUAUGCCUGAGAAUAUAUGAAGGAGGCUGGGAAGGCAAAGGGACGUUCAAUUG UCAUCACUGGC miR-205AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUG 251 UCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAU GGAGCUGACA miR-206-1UGCUUCCCGAGGCCACAUGCUUCUUUAUAUCCCC 252 AUAUGGAUUACUUUGCUAUGGAAUGUAAGGAAGUGUGUGGUUUCGGCAAGUG miR-206-2 AGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAU 253UACUUUGCUAUGGAAUGUAAGGAAGUGUGUGGU UUU miR-208UGACGGGCGAGCUUUUGGCCCGGGUUAUACCUG 254 AUGCUCACGUAUAAGACGAGCAAAAAGCUUGUUGGUCA miR-210 ACCCGGCAGUGCCUCCAGGCGCAGGGCAGCCCCU 255GCCCACCGCACACUGCGCUGCCCCAGACCCACUG UGCGUGUGACAGCGGCUGAUCUGUGCCUGGGCAGCGCGACCC miR-211 UCACCUGGCCAUGUGACUUGUGGGCUUCCCUUU 256GUCAUCCUUCGCCUAGGGCUCUGAGCAGGGCAG GGACAGCAAAGGGGUGCUCAGUUGUCACUUCCCACAGCACGGAG miR-212 CGGGGCACCCCGCCCGGACAGCGCGCCGGCACCU 257UGGCUCUAGACUGCUUACUGCCCGGGCCGCCCUC AGUAACAGUCUCCAGUCACGGCCACCGACGCCUGGCCCCGCC miR-213-2 CCUGUGCAGAGAUUAUUUUUUAAAAGGUCACAA 258UCAACAUUCAUUGCUGUCGGUGGGUUGAACUGU GUGGACAAGCUCACUGAACAAUGAAUGCAACUGUGGCCCCGCUU miR-213 GAGUUUUGAGGUUGCUUCAGUGAACAUUCAACG 259CUGUCGGUGAGUUUGGAAUUAAAAUCAAAACCA UCGACCGUUGAUUGUACCCUAUGGCUAACCAUCAUCUACUCC miR-214 GGCCUGGCUGGACAGAGUUGUCAUGUGUCUGCC 260UGUCUACACUUGCUGUGCAGAACAUCCGCUCAC CUGUACAGCAGGCACAGACAGGCAGUCACAUGACAACCCAGCCU miR-215 AUCAUUCAGAAAUGGUAUACAGGAAAAUGACCU 261AUGAAUUGACAGACAAUAUAGCUGAGUUUGUCU GUCAUUUCUUUAGGCCAAUAUUCUGUAUGACUGUGCUACUUCAA miR-216 GAUGGCUGUGAGUUGGCUUAAUCUCAGCUGGCA 262ACUGUGAGAUGUUCAUACAAUCCCUCACAGUGG UCUCUGGGAUUAUGCUAAACAGAGCAAUUUCCUAGCCCUCACGA miR-217 AGUAUAAUUAUUACAUAGUUUUUGAUGUCGCAG 263AUACUGCAUCAGGAACUGAUUGGAUAAGAAUCA GUCACCAUCAGUUCCUAAUGCAUUGCCUUCAGCAUCUAAACAAG miR-218-1 GUGAUAAUGUAGCGAGAUUUUCUGUUGUGCUUG 264AUCUAACCAUGUGGUUGCGAGGUAUGAGUAAAA CAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACA miR-218-2 GACCAGUCGCUGCGGGGCUUUCCUUUGUGCUUG 265AUCUAACCAUGUGGUGGAACGAUGGAAACGGAA CAUGGUUCUGUCAAGCACCGCGGAAAGCACCGUGCUCUCCUGCA miR-219 CCGCCCCGGGCCGCGGCUCCUGAUUGUCCAAACG 266CAAUUCUCGAGUCUAUGGCUCCGGCCGAGAGUU GAGUCUGGACGUCCCGAGCCGCCGCCCCCAAACCUCGAGCGGG miR-219-1 CCGCCCCGGGCCGCGGCUCCUGAUUGUCCAAACG 267CAAUUCUCGAGUCUAUGGCUCCGGCCGAGAGUU GAGUCUGGACGUCCCGAGCCGCCGCCCCCAAACCUCGAGCGGG miR-219-2 ACUCAGGGGCUUCGCCACUGAUUGUCCAAACGC 268AAUUCUUGUACGAGUCUGCGGCCAACCGAGAAU UGUGGCUGGACAUCUGUGGCUGAGCUCCGGGmiR-220 GACAGUGUGGCAUUGUAGGGCUCCACACCGUAU 269CUGACACUUUGGGCGAGGGCACCAUGCUGAAGG UGUUCAUGAUGCGGUCUGGGAACUCCUCACGGAUCUUACUGAUG miR-221 UGAACAUCCAGGUCUGGGGCAUGAACCUGGCAU 270ACAAUGUAGAUUUCUGUGUUCGUUAGGCAACAG CUACAUUGUCUGCUGGGUUUCAGGCUACCUGGAAACAUGUUCUC miR-222 GCUGCUGGAAGGUGUAGGUACCCUCAAUGGCUC 271AGUAGCCAGUGUAGAUCCUGUCUUUCGUAAUCA GCAGCUACAUCUGGCUACUGGGUCUCUGAUGGCAUCUUCUAGCU miR-223 CCUGGCCUCCUGCAGUGCCACGCUCCGUGUAUUU 272GACAAGCUGAGUUGGACACUCCAUGUGGUAGAG UGUCAGUUUGUCAAAUACCCCAAGUGCGGCACAUGCUUACCAG miR-224 GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUA 273GAUGAUUGUGCAUUGUUUCAAAAUGGUGCCCUA GUGACUACAAAGCCC miR-294-1CAAUCUUCCUUUAUCAUGGUAUUGAUUUUUCAG 274 (chr16)UGCUUCCCUUUUGUGUGAGAGAAGAUA miR-296 AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGU275 UGUGCCUAAUUCAGAGGGUUGGGUGGAGGCUCU CCUGAAGGGCUCU miR-299AAGAAAUGGUUUACCGUCCCACAUACAUUUUGA 276 AUAUGUAUGUGGGAUGGUAAACCGCUUCUUmiR-301 ACUGCUAACGAAUGCUCUGACUUUAUUGCACUA 277CUGUACUUUACAGCUAGCAGUGCAAUAGUAUUG UCAAAGCAUCUGAAAGCAGG miR-302aCCACCACUUAAACGUGGAUGUACUUGCUUUGAA 278 ACUAAAGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG miR-302b GCUCCCUUCAACUUUAACAUGGAAGUGCUUUCU 279GUGACUUUAAAAGUAAGUGCUUCCAUGUUUUAG UAGGAGU miR-302cCCUUUGCUUUAACAUGGGGGUACCUGCUGUGUG 280 AAACAAAAGUAAGUGCUUCCAUGUUUCAGUGGAGG miR-302d CCUCUACUUUAACAUGGAGGCACUUGCUGUGAC 281AUGACAAAAAUAAGUGCUUCCAUGUUUGAGUGU GG miR-320GCUUCGCUCCCCUCCGCCUUCUCUUCCCGGUUCU 282 UCCCGGAGUCGGGAAAAGCUGGGUUGAGAGGGCGAAAAAGGAUGAGGU miR-321 UUGGCCUCCUAAGCCAGGGAUUGUGGGUUCGAG 283UCCCACCCGGGGUAAAGAAAGGCCGA miR-323 UUGGUACUUGGAGAGAGGUGGUCCGUGGCGCGU 284UCGCUUUAUUUAUGGCGCACAUUACACGGUCGA CCUCUUUGCAGUAUCUAAUC miR-324CUGACUAUGCCUCCCCGCAUCCCCUAGGGCAUUG 285 GUGUAAAGCUGGAGACCCACUGCCCCAGGUGCUGCUGGGGGUUGUAGUC miR-325 AUACAGUGCUUGGUUCCUAGUAGGUGUCCAGUA 286AGUGUUUGUGACAUAAUUUGUUUAUUGAGGACC UCCUAUCAAUCAAGCACUGUGCUAGGCUCUGGmiR-326 CUCAUCUGUCUGUUGGGCUGGAGGCAGGGCCUU 287UGUGAAGGCGGGUGGUGCUCAGAUCGCCUCUGG GCCCUUCCUCCAGCCCCGAGGCGGAUUCA miR-328UGGAGUGGGGGGGCAGGAGGGGCUCAGGGAGAA 288 AGUGCAUACAGCCCCUGGCCCUCUCUGCCCUUCCGUCCCCUG miR-330 CUUUGGCGAUCACUGCCUCUCUGGGCCUGUGUC 289UUAGGCUCUGCAAGAUCAACCGAGCAAAGCACA CGGCCUGCAGAGAGGCAGCGCUCUGCCC miR-331GAGUUUGGUUUUGUUUGGGUUUGUUCUAGGUAU 290 GGUCCCAGGGAUCCCAGAUCAAACCAGGCCCCUGGGCCUAUCCUAGAACCAACCUAAGCUC miR-335 UGUUUUGAGCGGGGGUCAAGAGCAAUAACGAAA291 AAUGUUUGUCAUAAACCGUUUUUCAUUAUUGCU CCUGACCUCCUCUCAUUUGCUAUAUUCAmiR-337 GUAGUCAGUAGUUGGGGGGUGGGAACGGCUUCA 292UACAGGAGUUGAUGCACAGUUAUCCAGCUCCUA UAUGAUGCCUUUCUUCAUCCCCUUCAA miR-338UCUCCAACAAUAUCCUGGUGCUGAGUGAUGACU 293 CAGGCGACUCCAGCAUCAGUGAUUUUGUUGAAGAmiR-339 CGGGGCGGCCGCUCUCCCUGUCCUCCAGGAGCUC 294ACGUGUGCCUGCCUGUGAGCGCCUCGACGACAG AGCCGGCGCCUGCCCCAGUGUCUGCGC miR-340UUGUACCUGGUGUGAUUAUAAAGCAAUGAGACU 295 GAUUGUCAUAUGUCGUUUGUGGGAUCCGUCUCAGUUACUUUAUAGCCAUACCUGGUAUCUUA miR-342 GAAACUGGGCUCAAGGUGAGGGGUGCUAUCUGU296 GAUUGAGGGACAUGGUUAAUGGAAUUGUCUCAC ACAGAAAUCGCACCCGUCACCUUGGCCUACUUAmiR-345 ACCCAAACCCUAGGUCUGCUGACUCCUAGUCCAG 297GGCUCGUGAUGGCUGGUGGGCCCUGAACGAGGG GUCUGGAGGCCUGGGUUUGAAUAUCGACAGCmiR-346 GUCUGUCUGCCCGCAUGCCUGCCUCUCUGUUGCU 298CUGAAGGAGGCAGGGGCUGGGCCUGCAGCUGCC UGGGCAGAGCGGCUCCUGC miR-367CCAUUACUGUUGCUAAUAUGCAACUCUGUUGAA 299 UAUAAAUUGGAAUUGCACUUUAGCAAUGGUGAUGG miR-368 AAAAGGUGGAUAUUCCUUCUAUGUUUAUGUUAU 300UUAUGGUUAAACAUAGAGGAAAUUCCACGUUUU miR-369UUGAAGGGAGAUCGACCGUGUUAUAUUCGCUUU 301 AUUGACUUCGAAUAAUACAUGGUUGAUCUUUUCUCAG miR-370 AGACAGAGAAGCCAGGUCACGUCUCUGCAGUUA 302CACAGCUCACGAGUGCCUGCUGGGGUGGAACCU GGUCUGUCU miR-371GUGGCACUCAAACUGUGGGGGCACUUUCUGCUC 303 UCUGGUGAAAGUGCCGCCAUCUUUUGAGUGUUACmiR-372 GUGGGCCUCAAAUGUGGAGCACUAUUCUGAUGU 304CCAAGUGGAAAGUGCUGCGACAUUUGAGCGUCAC miR-373GGGAUACUCAAAAUGGGGGCGCUUUCCUUUUUG 305 UCUGUACUGGGAAGUGCUUCGAUUUUGGGGUGUCCC miR-374 UACAUCGGCCAUUAUAAUACAACCUGAUAAGUG 306UUAUAGCACUUAUCAGAUUGUAUUGUAAUUGUC UGUGUA miR-hes1AUGGAGCUGCUCACCCUGUGGGCCUCAAAUGUG 307 GAGGAACUAUUCUGAUGUCCAAGUGGAAAGUGCUGCGACAUUUGAGCGUCACCGGUGACGCCCAUA UCA miR-hes2GCAUCCCCUCAGCCUGUGGCACUCAAACUGUGG 308 GGGCACUUUCUGCUCUCUGGUGAAAGUGCCGCCAUCUUUUGAGUGUUACCGCUUGAGAAGACUCAA CC miR-hes3CGAGGAGCUCAUACUGGGAUACUCAAAAUGGGG 309 GCGCUUUCCUUUUUGUCUGUUACUGGGAAGUGCUUCGAUUUUGGGGUGUCCCUGUUUGAGUAGGGC AUC *An underlined sequence within aprecursor sequence corresponds to a mature processed miR transcript (seeTable 1b). Some precursor sequences have two underlined sequencesdenoting two different mature miRs that are derived from the sameprecursor. All sequences are human.

TABLE 1b Human Mature microRNA Sequences. Mature miRNA Mature miRNASequence SEQ ID Corresponding precursor Name (5′ to 3′) NO. microRNA(s);see Table 1a let-7a ugagguaguagguuguauaguu 310 let-7a-1; let-7a-2;let-7a-3; let-7a-4 let-7b ugagguaguagguugugugguu 311 let-7b let-7cugagguaguagguuguaugguu 312 let-7c let-7d agagguaguagguugcauagu 313let-7d; let-7d-v1 let-7e ugagguaggagguuguauagu 314 let-7e let-7fugagguaguagauuguauaguu 315 let-7f-1; let-7f-2-1; let-7f-2-2 let-7gugagguaguaguuuguacagu 316 let-7g let-7i ugagguaguaguuugugcu 317 let-7imiR-1 uggaauguaaagaaguaugua 318 miR-1b; miR-1b-1; miR-1b-2 miR-7uggaagacuagugauuuuguu 319 miR-7-1; miR-7-1a; miR-7-2; miR-7-3 miR-9ucuuugguuaucuagcuguauga 320 miR-9-1; miR-9-2; miR-9-3 miR-9*uaaagcuagauaaccgaaagu 321 miR-9-1; miR-9-2; miR-9-3 miR-10auacccuguagauccgaauuugug 322 miR-10a miR-10b uacccuguagaaccgaauuugu 323miR-10b miR-15a uagcagcacauaaugguuugug 324 miR-15a; miR-15a-2 miR-15buagcagcacaucaugguuuaca 325 miR-15b miR-16 uagcagcacguaaauauuggcg 326miR-16-1; miR-16-2; miR-16-13 miR-17- caaagugcuuacagugcagguagu 327miR-17 5p miR-17- acugcagugaaggcacuugu 328 miR-17 3p miR-18uaaggugcaucuagugcagaua 329 miR-18; miR-18-13 miR-19augugcaaaucuaugcaaaacuga 330 miR-19a; miR-19a-13 miR-19bugugcaaauccaugcaaaacuga 331 miR-19b-1; miR-19b-2 miR-20uaaagugcuuauagugcaggua 332 miR-20 (miR-20a) miR-21uagcuuaucagacugauguuga 333 miR-21; miR-21-17 miR-22aagcugccaguugaagaacugu 334 miR-22 miR-23a aucacauugccagggauuucc 335miR-23a miR-23b aucacauugccagggauuaccac 336 miR-23b miR-24uggcucaguucagcaggaacag 337 miR-24-1; miR-24-2; miR-24-19; miR-24-9miR-25 cauugcacuugucucggucuga 338 miR-25 miR-26a uucaaguaauccaggauaggcu339 miR-26a; miR-26a-1; miR-26a-2 miR-26b uucaaguaauucaggauaggu 340miR-26b miR-27a uucacaguggcuaaguuccgcc 341 miR-27a miR-27buucacaguggcuaaguucug 342 miR-27b-1; miR-27b-2 miR-28aaggagcucacagucuauugag 343 miR-28 miR-29a cuagcaccaucugaaaucgguu 344miR-29a-2; miR-29a miR-29b uagcaccauuugaaaucagu 345 miR-29b-1; miR-29b-2miR-29c uagcaccauuugaaaucgguua 346 miR-29c miR-30a-uguaaacauccucgacuggaagc 347 miR-30a 5p miR-30a- cuuucagucggauguuugcagc348 miR-30a 3p miR-30b uguaaacauccuacacucagc 349 miR-30b-1; miR-30b-2miR-30c uguaaacauccuacacucucagc 350 miR-30c miR-30duguaaacauccccgacuggaag 351 miR-30d miR-30e uguaaacauccuugacugga 352miR-30e miR-31 ggcaagaugcuggcauagcug 353 miR-31 miR-32uauugcacauuacuaaguugc 354 miR-32 miR-33 gugcauuguaguugcauug 355 miR-33;miR-33b miR-34a uggcagugucuuagcugguugu 356 miR-34a miR-34baggcagugucauuagcugauug 357 miR-34b miR-34c aggcaguguaguuagcugauug 358miR-34c miR-92 uauugcacuugucccggccugu 359 miR-92-2; miR-92-1 miR-93aaagugcuguucgugcagguag 360 miR-93-1; miR-93-2 miR-95uucaacggguauuuauugagca 361 miR-95 miR-96 uuuggcacuagcacauuuuugc 362miR-96 miR-98 ugagguaguaaguuguauuguu 363 miR-98 miR-99aaacccguagauccgaucuugug 364 miR-99a miR-99b cacccguagaaccgaccuugcg 365miR-99b miR-100 uacaguacugugauaacugaag 366 miR-100 miR-101uacaguacugugauaacugaag 367 miR-101-1; miR-101-2 miR-103agcagcauuguacagggcuauga 368 miR-103-1 miR-105 ucaaaugcucagacuccugu 369miR-105 miR-106-a aaaagugcuuacagugcagguagc 370 miR-106-a miR-106-buaaagugcugacagugcagau 371 miR-106-b miR-107 agcagcauuguacagggcuauca 372miR-107 miR-122a uggagugugacaaugguguuugu 373 miR-122a-1; miR-122a-2miR-124a uuaaggcacgcggugaaugcca 374 miR-124a-1; miR-124a-2; miR-124a-3miR-125a ucccugagacccuuuaaccugug 375 miR-125a-1; miR-125a-2 miR-125bucccugagacccuaacuuguga 376 miR-125b-1; miR-125b-2 miR-126*cauuauuacuuuugguacgcg 377 miR-126-1; miR-126-2 miR-126ucguaccgugaguaauaaugc 378 miR-126-1; miR-126-2 miR-127ucggauccgucugagcuuggcu 379 miR-127-1; miR-127-2 miR-128aucacagugaaccggucucuuuu 380 miR-128; miR-128a miR-128bucacagugaaccggucucuuuc 381 miR-128b miR-129 cuuuuugcggucugggcuugc 382miR-129-1; miR-129-2 miR-130a cagugcaauguuaaaagggc 383 miR-130a miR-130bcagugcaaugaugaaagggcau 384 miR-130b miR-132 uaacagucuacagccauggucg 385miR-132-1 miR-133a uugguccccuucaaccagcugu 386 miR-133a-1; miR-133a-2miR-133b uugguccccuucaaccagcua 387 miR-133b miR-134ugugacugguugaccagaggg 388 miR-134-1; miR-134-2 miR-135auauggcuuuuuauuccuauguga 389 miR-135a; miR-135a-2 (miR-135-2) miR-135buauggcuuuucauuccuaugug 390 miR-135b miR-136 acuccauuuguuuugaugaugga 391miR-136-1; miR-136-2 miR-137 uauugcuuaagaauacgcguag 392 miR-137 miR-138agcugguguugugaauc 393 miR-138-1; miR-138-2 miR-139 ucuacagugcacgugucu394 miR-139 miR-140 agugguuuuacccuaugguag 395 miR-140; miR-140as;miR-140s miR-141 aacacugucugguaaagaugg 396 miR-141-1; miR-141-2 miR-142-uguaguguuuccuacuuuaugga 397 miR-142 3p miR-142- cauaaaguagaaagcacuac 398miR-142 5p miR-143 ugagaugaagcacuguagcuca 399 miR-143-1 miR-144uacaguauagaugauguacuag 400 miR-144-1; miR-144-2 miR-145guccaguuuucccaggaaucccuu 401 miR-145-1; miR-145-2 miR-146ugagaacugaauuccauggguu 402 miR-146-1; miR-146-2 miR-147guguguggaaaugcuucugc 403 miR-147 miR-148a ucagugcacuacagaacuuugu 404miR-148a (miR-148) miR-148b ucagugcaucacagaacuuugu 405 miR-148b miR-149ucuggcuccgugucuucacucc 406 miR-149 miR-150 ucucccaacccuuguaccagug 407miR-150-1; miR-150-2 miR-151 acuagacugaagcuccuugagg 408 miR-151 miR-152ucagugcaugacagaacuugg 409 miR-152-1; miR-152-2 miR-153uugcauagucacaaaaguga 410 miR-153-1-1; miR-153-1- 2; miR-153-2-1;miR-153-2-2 miR-154 uagguuauccguguugccuucg 411 miR-154-1; miR-154-2miR-154* aaucauacacgguugaccuauu 412 miR-154-1; miR-154-2 miR-155uuaaugcuaaucgugauagggg 413 miR-155 miR-181a aacauucaacgcugucggugagu 414miR-181a miR-181b aacauucauugcugucgguggguu 415 miR-181b-1; miR-181b-2miR-181c aacauucaaccugucggugagu 416 miR-181c miR-182uuuggcaaugguagaacucaca 417 miR-182; miR-182as miR-182*ugguucuagacuugccaacua 418 miR-182; miR-182as miR-183uauggcacugguagaauucacug 419 miR-183 miR-184 uggacggagaacugauaagggu 420miR-184-1; miR-184-2 miR-185 uggagagaaaggcaguuc 421 miR-185-1; miR-185-2miR-186 caaagaauucuccuuuugggcuu 422 miR-186-1; miR-186-2 miR-187ucgugucuuguguugcagccg 423 miR-187 miR-188 caucccuugcaugguggagggu 424miR-188 miR-189 gugccuacugagcugauaucagu 425 miR-189-1; miR-189-2 miR-190ugauauguuugauauauuaggu 426 miR-190-1; miR-190-2 miR-191caacggaaucccaaaagcagcu 427 miR-191-1; miR-191-2 miR-192cugaccuaugaauugacagcc 428 miR-192 miR-193 aacuggccuacaaagucccag 429miR-193-1; miR-193-2 miR-194 uguaacagcaacuccaugugga 430 miR-194-1;miR-194-2 miR-195 uagcagcacagaaauauuggc 431 miR-195-1; miR-195-2miR-196a uagguaguuucauguuguugg 432 miR-196a; miR-196a-2 (miR196-2)miR-196b uagguaguuuccuguuguugg 433 miR-196b miR-197uucaccaccuucuccacccagc 434 miR-197 miR-198 gguccagaggggagauagg 435miR-198 miR-199a cccaguguucagacuaccuguuc 436 miR-199a-1; miR-199a-2 miR-uacaguagucugcacauugguu 437 miR-199a-1; miR-199a-2; 199a* miR-199s;miR-199b miR-199b cccaguguuuagacuaucuguuc 438 miR-199b miR-200auaacacugucugguaacgaugu 439 miR-200a miR-200b cucuaauacugccugguaaugaug440 miR-200b miR-200c aauacugccggguaaugaugga 441 miR-200c miR-202agagguauagggcaugggaaga 442 miR-202 miR-203 gugaaauguuuaggaccacuag 443miR-203 miR-204 uucccuuugucauccuaugccu 444 miR-204 miR-205uccuucauuccaccggagucug 445 miR-205 miR-206 uggaauguaaggaagugugugg 446miR-206-1; miR-206-2 miR-208 auaagacgagcaaaaagcuugu 447 miR-208 miR-210cugugcgugugacagcggcug 448 miR-210 miR-211 uucccuuugucauccuucgccu 449miR-211 miR-212 uaacagucuccagucacggcc 450 miR-212 miR-213accaucgaccguugauuguacc 451 miR-213 miR-214 acagcaggcacagacaggcag 452miR-214 miR-215 augaccuaugaauugacagac 453 miR-215 miR-216uaaucucagcuggcaacugug 454 miR-216 miR-217 uacugcaucaggaacugauuggau 455miR-217 miR-218 uugugcuugaucuaaccaugu 456 miR-218-1; miR-218-2 miR-219ugauuguccaaacgcaauucu 457 miR-219; miR-219-1; miR-219-2 miR-220ccacaccguaucugacacuuu 458 miR-220 miR-221 agcuacauugucugcuggguuuc 459miR-221 miR-222 agcuacaucuggcuacugggucuc 460 miR-222 miR-223ugucaguuugucaaauacccc 461 miR-223 miR-224 caagucacuagugguuccguuua 462miR-224 miR-296 agggcccccccucaauccugu 463 miR-296 miR-299ugguuuaccgucccacauacau 464 miR-299 miR-301 cagugcaauaguauugucaaagc 465miR-301 miR-302a uaagugcuuccauguuuugguga 466 miR-302a miR-acuuuaacauggaagugcuuucu 467 miR-302b 302b* miR-302buaagugcuuccauguuuuaguag 468 miR-302b miR- uuuaacauggggguaccugcug 469miR-302c 302c* miR-302c uaagugcuuccauguuucagugg 470 miR-302c miR-302duaagugcuuccauguuugagugu 471 miR-302d miR-320 aaaagcuggguugagagggcgaa 472miR-320 miR-321 uaagccagggauuguggguuc 473 miR-321 miR-323gcacauuacacggucgaccucu 474 miR-323 miR-324- cgcauccccuagggcauuggugu 475miR-324 5p miR-324- ccacugccccaggugcugcugg 476 miR-324 3p miR-325ccuaguagguguccaguaagu 477 miR-325 miR-326 ccucugggcccuuccuccag 478miR-326 miR-328 cuggcccucucugcccuuccgu 479 miR-328 miR-330gcaaagcacacggccugcagaga 480 miR-330 miR-331 gccccugggccuauccuagaa 481miR-331 miR-335 ucaagagcaauaacgaaaaaugu 482 miR-335 miR-337uccagcuccuauaugaugccuuu 483 miR-337 miR-338 uccagcaucagugauuuuguuga 484miR-338 miR-339 ucccuguccuccaggagcuca 485 miR-339 miR-340uccgucucaguuacuuuauagcc 486 miR-340 miR-342 ucucacacagaaaucgcacccguc 487miR-342 miR-345 ugcugacuccuaguccagggc 488 miR-345 miR-346ugucugcccgcaugccugccucu 489 miR-346 miR-367 aauugcacuuuagcaaugguga 490miR-367 miR-368 acauagaggaaauuccacguuu 491 miR-368 miR-369aauaauacaugguugaucuuu 492 miR-369 miR-370 gccugcugggguggaaccugg 493miR-370 miR-371 gugccgccaucuuuugagugu 494 miR-371 miR-372aaagugcugcgacauuugagcgu 495 miR-372 miR-373* acucaaaaugggggcgcuuucc 496miR-373 miR-373 gaagugcuucgauuuuggggugu 497 miR-373 miR-374uuauaauacaaccugauaagug 498 miR-374

The present invention encompasses methods of diagnosing whether asubject has, or is at risk for developing, a solid cancer, comprisingmeasuring the level of at least one miR gene product in a test samplefrom the subject and comparing the level of the miR gene product in thetest sample to the level of a corresponding miR gene product in acontrol sample. As used herein, a “subject” can be any mammal that has,or is suspected of having, a solid cancer. In a preferred embodiment,the subject is a human who has, or is suspected of having, a solidcancer.

In one embodiment, the at least one miR gene product measured in thetest sample is selected from the group consisting of miR-21, miR-17-5p,miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2,miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2,miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof. Ina particular embodiment, the miR gene product is miR-21, miR-191 ormiR-17-5p. In another embodiment, the miR gene product is not miR-15a ormiR-16-1. In an additional embodiment, the miR gene product is not miR159-1 or miR-192. In an additional embodiment, the miR gene product isnot miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93,miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183, miR-129-1,let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32,miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b,miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175.In a further embodiment, the miR gene product is not miR-21, miR-301,miR-142 as, miR-142s, miR-194, miR-215, or miR-32. In anotherembodiment, the miR gene product is not miR-148, miR-10a, miR-196-1,miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202,miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the miRgene product is not miR-15a, miR-16-1, miR-182, miR-181, miR-30,miR-15a, miR-16-1, miR-15b, miR-16-2, miR-195, miR-34, miR-153, miR-21,miR-217, miR-205, miR-204, miR-211, miR-143, miR-96, miR-103, miR-107,miR-129, miR-9, miR-137, miR-217, miR-186.

The solid cancer can be any cancer that arises from organs and solidtissues. Such cancers are typically associated with the formation and/orpresence of tumor masses and can be carcinomas, sarcomas and lymphomas.Specific examples of solid cancers to be diagnosed by the methods of theinvention include, but are not limited to, colon cancer, rectal cancer,stomach (gastric) cancer, pancreatic cancer, breast cancer, lung cancer,prostate cancer, bronchial cancer, testicular cancer, ovarian cancer,uterine cancer, penile cancer, melanoma and other skin cancers, livercancer, esophogeal cancer, cancers of the oral cavity and pharynx (e.g.,tongue cancer, mouth cancer), cancers of the digestive system (e.g.,intestinal cancer, gall bladder cancer), bone and joint cancers, cancersof the endocrine system (e.g., thyroid cancer), brain cancer, eyecancer, cancers of the urinary system (e.g., kidney cancer, urinarybladder cancer), Hodgkin disease and non-Hodgkin lymphoma. In particularembodiments, the solid cancer is not one or more of breast cancer, lungcancer, prostate cancer, pancreatic cancer or gastrointestinal cancer.

In one embodiment, the solid cancer is breast cancer or lung cancer andthe at least one miR gene product measured in the test sample isselected from the group consisting of miR-210, miR-213 and a combinationthereof.

In a further embodiment, the solid cancer is colon cancer, stomachcancer, prostate cancer or pancreas cancer and the at least one miR geneproduct measured in the test sample is miR-218-2.

In a certain embodiment of the invention, the solid cancer is breastcancer and the at least one miR gene product measured in the test sampleis selected from the group consisting of miR-125b-1, miR-125b-2,miR-145, miR-21 and combinations thereof. In a related embodiment, thesolid cancer is breast cancer and the at least one miR gene product inthe test sample is selected from the group consisting of miR-21,miR-29b-2, miR-146, miR-125b-2, miR-125b-1, miR-10b, miR-145, miR-181a,miR-140, miR-213, miR-29a prec, miR-181b-1, miR-199b, miR-29b-1,miR-130a, miR-155, let-7a-2, miR-205, miR-29c, miR-224, miR-100, miR-31,miR-30c, miR-17-5p, miR-210, miR-122a, miR-16-2 and combinationsthereof. In a related embodiment, the solid cancer is breast cancer andthe at least one miR gene product is not miR-15a or miR-16-1. In afurther embodiment, the solid cancer is breast cancer and the at leastone miR gene product is not miR-145, miR-21, miR-155, miR-10b,miR-125b-1, miR-125b-2, let7a-2, let7a-3, let-7d, miR-122a, miR-191,miR-206, miR-210, let-71, miR-009-1 (miR131-1), miR-34 (miR-170),miR-102 (miR-29b), miR-123 (miR-126), miR-140-as, miR-125a, miR-194,miR-204, miR-213, let-7f-2, miR-101, miR-128b, miR-136, miR-143,miR-149, miR-191, miR-196-1, miR-196-2, miR-202, miR-103-1, or miR-30c.In another embodiment, the solid cancer is breast cancer and the miRgene product is not miR-21, miR-125b-1, let-7a-2, let-71, miR-100,let-7g, miR-31, miR-32a-1, miR-33b, miR-34a-2, miR-101-1, miR-142 as,miR-142s, miR-301, miR-29c, miR-30c, miR-106a, or miR-29b-1. In yetanother embodiment, the solid cancer is breast cancer and the miR geneproduct is not miR-159-1 or miR-192. In an additional embodiment, thesolid cancer is breast cancer and the miR gene product is not miR-186,miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a,miR-96, miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d,let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192,miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21,miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175. In a furtherembodiment, the solid cancer is breast cancer and the miR gene productis not miR-21, miR-301, miR-142 as, miR-142s, miR-194, miR-215, ormiR-32. In another embodiment, the solid cancer is breast cancer and themiR gene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2,miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a,let-7f, or let-7d. In yet another embodiment, the solid cancer is breastcancer and the miR gene product is not miR-181b, miR-181c, miR-181d,miR-30, miR-15b, miR-16-2, miR-153-1, miR-217, miR-205, miR-204,miR-103, miR-107, miR-129-2, miR-9 or miR-137.

In another embodiment, the solid cancer is colon cancer and the at leastone miR gene product in the test sample is selected from the groupconsisting of miR-24-1, miR-29b-2, miR-20a, miR-10a, miR-32, miR-203,miR-106a, miR-17-5p, miR-30c, miR-223, miR-126*, miR-128b, miR-21,miR-24-2, miR-99b prec, miR-155, miR-213, miR-150, miR-107, miR-191,miR-221, miR-9-3 and combinations thereof. In another embodiment, thesolid cancer is colon cancer and the miR gene product is not miR 159-1or miR-192. In an additional embodiment, the solid cancer is coloncancer and the miR gene product is not miR-186, miR-101-1, miR-194,miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s,miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b,miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2,miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s,miR-142 as, miR-105-1, or miR-175. In a further embodiment, the solidcancer is colon cancer and the miR gene product is not miR-21, miR-301,miR-142 as, miR-142s, miR-194, miR-215, or miR-32. In anotherembodiment, the solid cancer is colon cancer and the miR gene product isnot miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b,miR-129-1, miR-153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. Inyet another embodiment, the solid cancer is colon cancer and the miRgene product is not miR-181b, miR-181c, miR-181d, miR-30, miR-15b,miR-16-2, miR-153-1, miR-217, miR-205, miR-204, miR-103, miR-107,miR-129-2, miR-9 or miR-137.

In yet another embodiment, the solid cancer is lung cancer and the miRgene product in the test sample is selected from the group consisting ofmiR-21, miR-205, miR-200b, miR-9-1, miR-210, miR-148, miR-141, miR-132,miR-215, miR-128b, let-7g, miR-16-2, miR-129-1/2 prec, miR-126*,miR-142-as, miR-30d, miR-30a-5p, miR-7-2, miR-199a-1, miR-127, miR-34aprec, miR-34a, miR-136, miR-202, miR-196-2, miR-199a-2, let-7a-2,miR-124a-1, miR-149, miR-17-5p, miR-196-1 prec, miR-10a, miR-99b prec,miR-196-1, miR-199b, miR-191, miR-195, miR-155 and combinations thereof.In a related embodiment, the solid cancer is lung cancer and the atleast one miR gene product is not miR-15a or miR-16-1. In a furtherembodiment, the solid cancer is lung cancer and the at least one miRgene product is not miR-21, miR-191, miR-126*, miR-210, miR-155,miR-143, miR-205, miR-126, miR-30a-5p, miR-140, miR-214, miR-218-2,miR-145, miR-106a, miR-192, miR-203, miR-150, miR-220, miR-192, miR-224,miR-24-2, miR-212, miR-9, miR-17, miR-124a-1, miR-95, miR-198, miR-216,miR-219-1, miR-197, miR-125a, miR-26a-1, miR-146, miR-199b, let7a-2,miR-27b, miR-32, miR-29b-2, miR-33, miR-181c, miR-101-1, miR-124a-3,miR-125b-1 or let7f-1. In another embodiment, the solid cancer is lungcancer and the at least one miR gene product is not miR-21, miR-182,miR-181, miR-30, miR-15a, miR-143, miR-205, miR-96, miR-103, miR-107,miR-129, miR-137, miR-186, miR-15b, miR-16-2, miR-195, miR-34, miR-153,miR-217, miR-204, miR-211, miR-9, miR-217, let-7a-2 or miR-32. In afurther embodiment, the solid cancer is lung cancer and the miR geneproduct is not let-7c, let-7g, miR-7-3, miR-210, miR-31, miR-34a-1,miR-a-2, miR-99a, miR-100, miR-125b-2, miR-132, miR-135-1, miR-195,miR-34, miR-123, miR-203. In another embodiment, the solid cancer islung cancer and the miR gene product is not miR 159-1 or miR-192. In anadditional embodiment, the solid cancer is lung cancer and the miR geneproduct is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25,miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183,miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b,miR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2,miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, ormiR-175. In a further embodiment, the solid cancer is lung cancer andthe miR gene product is not miR-21, miR-301, miR-142 as, miR-142s,miR-194, miR-215, or miR-32. In another embodiment, the solid cancer islung cancer and the miR gene product is not miR-148, miR-10a, miR-196-1,miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202,miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the solidcancer is lung cancer and the miR gene product is not miR-181b,miR-181c, miR-181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217,miR-205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.

In a further embodiment, the solid cancer is pancreatic cancer and theat least one miR gene product measured in the test sample is selectedfrom the group consisting of miR-103-1, miR-103-2, miR-155, miR-204 andcombinations thereof. In a related embodiment, the solid cancer ispancreatic cancer and the miR gene product in the test sample isselected from the group consisting of miR-103-2, miR-103-1, miR-24-2,miR-107, miR-100, miR-125b-2, miR-125b-1, miR-24-1, miR-191, miR-23a,miR-26a-1, miR-125a, miR-130a, miR-26b, miR-145, miR-221, miR-126*,miR-16-2, miR-146, miR-214, miR-99b, miR-128b, miR-155, miR-29b-2,miR-29a, miR-25, miR-16-1, miR-99a, miR-224, miR-30d, miR-92-2,miR-199a-1, miR-223, miR-29c, miR-30b, miR-129-1/2, miR-197, miR-17-5p,miR-30c, miR-7-1, miR-93-1, miR-140, miR-30a-5p, miR-132, miR-181b-1,miR-152 prec, miR-23b, miR-20a, miR-222, miR-27a, miR-92-1, miR-21,miR-129-1/2 prec, miR-150, miR-32, miR-106a, miR-29b-1 and combinationsthereof. In one embodiment, the solid cancer is pancreatic cancer andthe miR gene product is not miR-15a or miR-16-1. In another embodiment,the solid cancer is pancreatic cancer and the miR gene product is notmiR 159-1 or miR-192. In an additional embodiment, the solid cancer ispancreatic cancer and the miR gene product is not miR-186, miR-101-1,miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96,miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1,miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-1,let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301,miR-142s, miR-142 as, miR-105-1, or miR-175. In a further embodiment,the solid cancer is pancreatic cancer and the miR gene product is notmiR-21, miR-301, miR-142 as, miR-1425, miR-194, miR-215, or miR-32. Inanother embodiment, the solid cancer is pancreatic cancer and the miRgene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2,miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a,let-7f, or let-7d. In yet another embodiment, the solid cancer ispancreatic cancer and the miR gene product is not miR-181b, miR-181c,miR-181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217, miR-205,miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.

In another embodiment, the solid cancer is prostate cancer and the miRgene product in the test sample is selected from the group consisting oflet-7d, miR-128a prec, miR-195, miR-203, let-7a-2 prec, miR-34a,miR-20a, miR-218-2, miR-29a, miR-25, miR-95, miR-197, miR-135-2,miR-187, miR-196-1, miR-148, miR-191, miR-21, let-71, miR-198,miR-199a-2, miR-30c, miR-17-5p, miR-92-2, miR-146, miR-181b-1 prec,miR-32, miR-206, miR-184 prec, miR-29a prec, miR-29b-2, miR-149,miR-181b-1, miR-196-1 prec, miR-93-1, miR-223, miR-16-1, miR-101-1,miR-124a-1, miR-26a-1, miR-214, miR-27a, miR-24-1, miR-106a, miR-199a-1and combinations thereof. In a related embodiment, the solid cancer isprostate cancer and the miR gene product is not miR-15a or miR-16-1. Inanother embodiment, the solid cancer is prostate cancer and the miR geneproduct is not miR 159-1 or miR-192. In an additional embodiment, thesolid cancer is prostate cancer and the miR gene product is not miR-186,miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a,miR-96, miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d,let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192,miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21,miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175. In a furtherembodiment, the solid cancer is prostate cancer and the miR gene productis not miR-21, miR-301, miR-142 as, miR-142s, miR-194, miR-215, ormiR-32.

In another embodiment, the solid cancer is prostate cancer and the miRgene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2,miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a,let-7f, or let-7d. In yet another embodiment, the solid cancer isprostate cancer and the miR gene product is not miR-181b, miR-181c,miR-181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217, miR-205,miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.

In yet another embodiment, the solid Cancer is stomach cancer and themiR gene product in the test sample is selected from the groupconsisting of miR-223, miR-21, miR-218-2, miR-103-2, miR-92-2, miR-25,miR-136, miR-191, miR-221, miR-125b-2, miR-103-1, miR-214, miR-222,miR-212 prec, miR-125b-1, miR-100, miR-107, miR-92-1, miR-96, miR-192,miR-23a, miR-215, miR-7-2, miR-138-2, miR-24-1, miR-99b, miR-33b,miR-24-2 and combinations thereof. In a related embodiment, the solidcancer is stomach cancer and the miR gene product is not miR-15a ormiR-16-1. In another embodiment, the solid cancer is stomach cancer andthe miR gene product is not miR 159-1 or miR-192. In an additionalembodiment, the solid cancer is stomach cancer and the miR gene productis not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93,miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183, miR-129-1,let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32,miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b,miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175.In a further embodiment, the solid cancer is stomach cancer and the miRgene product is not miR-21, miR-301, miR-142a5, miR-142s, miR-194,miR-215, or miR-32. In another embodiment, the solid cancer is stomachcancer and the miR gene product is not miR-148, miR-10a, miR-196-1,miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202,miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the solidcancer is stomach cancer and the miR gene product is not miR-181b,miR-181c, miR-181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217,miR-205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.

The level of at least one miR gene product can be measured in abiological sample (e.g., cells, tissues) obtained from the subject. Forexample, a tissue sample (e.g., from a tumor) can be removed from asubject suspected of having a solid cancer by conventional biopsytechniques. In another embodiment, a blood sample can be removed fromthe subject, and blood cells (e.g., white blood cells) can be isolatedfor DNA extraction by standard techniques. The blood or tissue sample ispreferably obtained from the subject prior to initiation ofradiotherapy, chemotherapy or other therapeutic treatment. Acorresponding control tissue or blood sample can be obtained fromunaffected tissues of the subject, from a normal human individual orpopulation of normal individuals, or from cultured cells correspondingto the majority of cells in the subject's sample. The control tissue orblood sample is then processed along with the sample from the subject,so that the levels of miR gene product produced from a given miR gene incells from the subject's sample can be compared to the corresponding miRgene product levels from cells of the control sample. A reference miRexpression standard for the biological sample can also be used as acontrol.

An alteration (e.g., an increase or decrease) in the level of a miR geneproduct in the sample obtained from the subject, relative to the levelof a corresponding miR gene product in a control sample, is indicativeof the presence of a solid cancer in the subject. In one embodiment, thelevel of the at least one miR gene product in the test sample is greaterthan the level of the corresponding miR gene product in the controlsample (i.e., expression of the miR gene product is “up-regulated”). Asused herein, expression of a miR gene product is “up-regulated” when theamount of miR gene product in a cell or tissue sample from a subject isgreater than the amount of the same gene product in a control cell ortissue sample. In another embodiment, the level of the at least one miRgene product in the test sample is less than the level of thecorresponding miR gene product in the control sample (i.e., expressionof the miR gene product is “down-regulated”). As used herein, expressionof a miR gene is “down-regulated” when the amount of miR gene productproduced from that gene in a cell or tissue sample from a subject isless than the amount produced from the same gene in a control cell ortissue sample. The relative miR gene expression in the control andnormal samples can be determined with respect to one or more RNAexpression standards. The standards can comprise, for example, a zeromiR gene expression level, the miR gene expression level in a standardcell line, the miR gene expression level in unaffected tissues of thesubject, or the average level of miR gene expression previously obtainedfor a population of normal human controls.

The level of a miR gene product in a sample can be measured using anytechnique that is suitable for detecting RNA expression levels in abiological sample. Suitable techniques (e.g., Northern blot analysis,RT-PCR, in situ hybridization) for determining RNA expression levels ina biological sample (e.g., cells, tissues) are well known to those ofskill in the art. In a particular embodiment, the level of at least onemiR gene product is detected using Northern blot analysis. For example,total cellular RNA can be purified from cells by homogenization in thepresence of nucleic acid extraction buffer, followed by centrifugation.Nucleic acids are precipitated, and DNA is removed by treatment withDNase and, precipitation. The RNA molecules are then separated by gelelectrophoresis on agarose gels according to standard techniques, andtransferred to nitrocellulose filters. The RNA is then immobilized onthe filters by heating. Detection and quantification of specific RNA isaccomplished using appropriately labeled DNA or RNA probes complementaryto the RNA in question. See, for example, Molecular Cloning: ALaboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold SpringHarbor Laboratory Press, 1989, Chapter 7, the entire disclosure of whichis incorporated by reference.

Suitable probes for Northern blot hybridization of a given miR geneproduct can be produced from the nucleic acid sequences provided inTable 1a and Table 1b and include, but are not limited to, probes havingat least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or completecomplementarity to a miR gene product of interest. Methods forpreparation of labeled DNA and RNA probes, and the conditions forhybridization thereof to target nucleotide sequences, are described inMolecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2ndedition, Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11,the disclosures of which are incorporated herein by reference.

For example, the nucleic acid probe can be labeled with, e.g., aradionuclide, such as ³H, ³²P, ³³P, ¹⁴C, or ³⁵S; a heavy metal; a ligandcapable of functioning as a specific binding pair member for a labeledligand (e.g., biotin, avidin or an antibody); a fluorescent molecule; achemiluminescent molecule; an enzyme or the like.

Probes can be labeled to high specific activity by either the nicktranslation method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 orby the random priming method of Fienberg et al. (1983), Anal. Biochem.132:6-13, the entire disclosures of which are incorporated herein byreference. The latter is the method of choice for synthesizing³²P-labeled probes of high specific activity from single-stranded DNA orfrom RNA templates. For example, by replacing preexisting nucleotideswith highly radioactive nucleotides according to the nick translationmethod, it is possible to prepare ³²P-labeled nucleic acid probes with aspecific activity well in excess of 10⁸ cpm/microgram. Autoradiographicdetection of hybridization can then be performed by exposing hybridizedfilters to photographic film. Densitometric scanning of the photographicfilms exposed by the hybridized filters provides an accurate measurementof miR gene transcript levels. Using another approach, miR genetranscript levels can be quantified by computerized imaging systems,such as the Molecular Dynamics 400-B 2D Phosphorimager available fromAmersham Biosciences, Piscataway, N.J.

Where radionuclide labeling of DNA or RNA probes is not practical, therandom-primer method can be used to incorporate an analogue, forexample, the dTTP analogue5-(N-(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridinetriphosphate, into the probe molecule. The biotinylated probeoligonucleotide can be detected by reaction with biotin-bindingproteins, such as avidin, streptavidin, and antibodies (e.g.,anti-biotin antibodies) coupled to fluorescent dyes or enzymes thatproduce color reactions.

In addition to Northern and other RNA hybridization techniques,determining the levels of RNA transcripts can be accomplished using thetechnique of in situ hybridization. This technique requires fewer cellsthan the Northern blotting technique, and involves depositing wholecells onto a microscope cover slip and probing the nucleic acid contentof the cell with a solution containing radioactive or otherwise labelednucleic acid (e.g., cDNA or RNA) probes. This technique is particularlywell-suited for analyzing tissue biopsy samples from subjects. Thepractice of the in situ hybridization technique is described in moredetail in U.S. Pat. No. 5,427,916, the entire disclosure of which isincorporated herein by reference. Suitable probes for in situhybridization of a given miR gene product can be produced from thenucleic acid sequences provided in Table 1a and Table 1b, and include,but are not limited to, probes having at least about 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or complete complementarity to a miR gene product ofinterest, as described above.

The relative number of miR gene transcripts in cells can also bedetermined by reverse transcription of miR gene transcripts, followed byamplification of the reverse-transcribed transcripts by polymerase chainreaction (RT-PCR). The levels of miR gene transcripts can be quantifiedin comparison with an internal standard, for example, the level of mRNAfrom a “housekeeping” gene present in the same sample. A suitable“housekeeping” gene for use as an internal standard includes, e.g.,myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Methods forperforming quantitative and semi-quantitative RT-PCR, and variationsthereof, are well known to those of skill in the art.

In some instances, it may be desirable to simultaneously determine theexpression level of a plurality of different miR gene products in asample. In other instances, it may be desirable to determine theexpression level of the transcripts of all known miR genes correlatedwith a cancer. Assessing cancer-specific expression levels for hundredsof miR genes or gene products is time consuming and requires a largeamount of total RNA (e.g., at least 20 μg for each Northern blot) andautoradiographic techniques that require radioactive isotopes.

To overcome these limitations, an oligolibrary, in microchip format(i.e., a microarray), may be constructed containing a set ofoligonucleotide (e.g., oligodeoxynucleotides) probes that are specificfor a set of miR genes. Using such a microarray, the expression level ofmultiple microRNAs in a biological sample can be determined by reversetranscribing the RNAs to generate a set of target oligodeoxynucleotides,and hybridizing them to probe the oligonucleotides on the microarray togenerate a hybridization, or expression, profile. The hybridizationprofile of the test sample can then be compared to that of a controlsample to determine which microRNAs have an altered expression level insolid cancer cells. As used herein, “probe oligonucleotide” or “probeoligodeoxynucleotide” refers to an oligonucleotide that is capable ofhybridizing to a target oligonucleotide. “Target oligonucleotide” or“target oligodeoxynucleotide” refers to a molecule to be detected (e.g.,via hybridization). By “miR-specific probe oligonucleotide” or “probeoligonucleotide specific for a miR” is meant a probe oligonucleotidethat has a sequence selected to hybridize to a specific miR geneproduct, or to a reverse transcript of the specific miR gene product.

An “expression profile” or “hybridization profile” of a particularsample is essentially a fingerprint of the state of the sample; whiletwo states may have any particular gene similarly expressed, theevaluation of a number of genes simultaneously allows the generation ofa gene expression profile that is unique to the state of the cell. Thatis, normal tissue may be distinguished from cancerous (e.g., tumor)tissue, and within cancerous tissue, different prognosis states (forexample, good or poor long term survival prospects) may be determined.By comparing expression profiles of solid cancer tissue in differentstates, information regarding which genes are important (including bothup- and down-regulation of genes) in each of these states is obtained.The identification of sequences that are differentially expressed insolid cancer tissue, as well as differential expression resulting indifferent prognostic outcomes, allows the use of this information in anumber of ways. For example, a particular treatment regime may beevaluated (e.g., to determine whether a chemotherapeutic drug acts toimprove the long-term prognosis in a particular patient). Similarly,diagnosis may be done or confirmed by comparing patient samples withknown expression profiles. Furthermore, these gene expression profiles(or individual genes) allow screening of drug candidates that suppressthe solid cancer expression profile or convert a poor prognosis profileto a better prognosis profile.

Accordingly, the invention provides methods of diagnosing whether asubject has, or is at risk for developing, a solid cancer, comprisingreverse transcribing RNA from a test sample obtained from the subject toprovide a set of target oligodeoxynucleotides, hybridizing the targetoligodeoxynucleotides to a microarray comprising miRNA-specific probeoligonucleotides to provide a hybridization profile for the test sample,and comparing the test sample hybridization profile to a hybridizationprofile generated from a control sample or reference standard, whereinan alteration in the signal of at least one miRNA is indicative of thesubject either having, or being at risk for developing, a solid cancer.In one embodiment, the microarray comprises miRNA-specific probeoligonucleotides for a substantial portion of all known human miRNAs. Ina particular embodiment, the microarray comprises miRNA-specific probeoligonucleotides for one or more miRNAs selected from the groupconsisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b,miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a,miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106aand combinations thereof.

The microarray can be prepared from gene-specific oligonucleotide probesgenerated from known miRNA sequences. The array may contain twodifferent oligonucleotide probes for each miRNA, one containing theactive, mature sequence and the other being specific for the precursorof the miRNA. The array may also contain controls, such as one or moremouse sequences differing from human orthologs by only a few bases,which can serve as controls for hybridization stringency conditions.tRNAs or other RNAs (e.g., rRNAs, mRNAs) from both species may also beprinted on the microchip, providing an internal, relatively stable,positive control for specific hybridization. One or more appropriatecontrols for non-specific hybridization may also be included on themicrochip. For this purpose, sequences are selected based upon theabsence of any homology with any known miRNAs.

The microarray may be fabricated using techniques known in the art. Forexample, probe oligonucleotides of an appropriate length, e.g., 40nucleotides, are 5′-amine modified at position C6 and printed usingcommercially available microarray systems, e.g., the GeneMachineOmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides.Labeled cDNA oligomer corresponding to the target RNAs is prepared byreverse transcribing the target RNA with labeled primer. Following firststrand synthesis, the RNA/DNA hybrids are denatured to degrade the RNAtemplates. The labeled target cDNAs thus prepared are then hybridized tothe microarray chip under hybridizing conditions, e.g., 6×SSPE/30%formamide at 25° C. for 18 hours, followed by washing in 0.75×TNT (TrisHCl/NaCl/Tween 20) at 37° C. for 40 minutes. At positions on the arraywhere the immobilized probe DNA recognizes a complementary target cDNAin the sample, hybridization occurs. The labeled target cDNA marks theexact position on the array where binding occurs, allowing automaticdetection and quantification. The output consists of a list ofhybridization events, indicating the relative abundance of specific cDNAsequences, and therefore the relative abundance of the correspondingcomplementary miRs, in the patient sample. According to one embodiment,the labeled cDNA oligomer is a biotin-labeled cDNA, prepared from abiotin-labeled primer. The microarray is then processed by directdetection of the biotin-containing transcripts using, e.g.,Streptavidin-Alexa647 conjugate, and scanned utilizing conventionalscanning methods. Image intensities of each spot on the array areproportional to the abundance of the corresponding miR in the patientsample.

The use of the array has several advantages for miRNA expressiondetection. First, the global expression of several hundred genes can beidentified in the same sample at one time point. Second, through carefuldesign of the oligonucleotide probes, expression of both mature andprecursor molecules can be identified. Third, in comparison withNorthern blot analysis, the chip requires a small amount of RNA, andprovides reproducible results using 2.5 μg of total RNA. The relativelylimited number of miRNAs (a few hundred per species) allows theconstruction of a common microarray for several species, with distinctoligonucleotide probes for each. Such a tool would allow for analysis oftrans-species expression for each known miR under various conditions.

In addition to use for quantitative expression level assays of specificmiRs, a microchip containing miRNA-specific probe oligonucleotidescorresponding to a substantial portion of the miRNome, preferably theentire miRNome, may be employed to carry out miR gene expressionprofiling, for analysis of miR expression patterns. Distinct miRsignatures can be associated with established disease markers, ordirectly with a disease state.

According to the expression profiling methods described herein, totalRNA from a sample from a subject suspected of having a cancer (e.g., asolid cancer) is quantitatively reverse transcribed to provide a set oflabeled target oligodeoxynucleotides complementary to the RNA in thesample. The target oligodeoxynucleotides are then hybridized to amicroarray comprising miRNA-specific probe oligonucleotides to provide ahybridization profile for the sample. The result is a hybridizationprofile for the sample representing the expression pattern of miRNA inthe sample. The hybridization profile comprises the signal from thebinding of the target oligodeoxynucleotides from the sample to themiRNA-specific probe oligonucleotides in the microarray. The profile maybe recorded as the presence or absence of binding (signal vs. zerosignal). More preferably, the profile recorded includes the intensity ofthe signal from each hybridization. The profile is compared to thehybridization profile generated from a normal, i.e., noncancerous,control sample. An alteration in the signal is indicative of thepresence of, or propensity to develop, cancer in the subject.

Other techniques for measuring miR gene expression are also within theskill in the art, and include various techniques for measuring rates ofRNA transcription and degradation.

The invention also provides methods of determining the prognosis of asubject with a solid cancer, comprising measuring the level of at leastone miR gene product, which is associated with a particular prognosis ina solid cancer (e.g., a good or positive prognosis, a poor or adverseprognosis), in a test sample from the subject. According to thesemethods, an alteration in the level of a miR gene product that isassociated with a particular prognosis in the test sample, as comparedto the level of a corresponding miR gene product in a control sample, isindicative of the subject having a solid cancer with a particularprognosis. In one embodiment, the miR gene product is associated with anadverse (i.e., poor) prognosis. Examples of an adverse prognosisinclude, but are not limited to, low survival rate and rapid diseaseprogression. In certain embodiments, the level of the at least one miRgene product is measured by reverse transcribing RNA from a test sampleobtained from the subject to provide a set of targetoligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to amicroarray that comprises miRNA-specific probe oligonucleotides toprovide a hybridization profile for the test sample, and comparing thetest sample hybridization profile to a hybridization profile generatedfrom a control sample.

Without wishing to be bound by any one theory, it is believed thatalterations in the level of one or more miR gene products in cells canresult in the deregulation of one or more intended targets for thesemiRs, which can lead to the formation of solid cancers. Therefore,altering the level of the miR gene product (e.g., by decreasing thelevel of a miR gene product that is up-regulated in solid cancer cells,by increasing the level of a miR gene product that is down-regulated insolid cancer cells) may successfully treat the solid cancer.

Accordingly, the present invention encompasses methods of inhibitingtumorigenesis in a subject who has, or is suspected of having, a solidcancer wherein at least one miR gene product is deregulated (e.g.,down-regulated, up-regulated) in the cancer cells of the subject. Whenthe at least one isolated miR gene product is down-regulated in thecancer cells (e.g., miR-145, miR-155, miR-218-2), the method comprisesadministering an effective amount of the at least one isolated miR geneproduct, or an isolated variant or biologically-active fragment thereof,such that proliferation of cancer cells in the subject is inhibited. Inone embodiment, the isolated miR gene product that is administered isnot miR-15a or miR-16-1. In another embodiment, the miR gene product isnot miR 159-1 or miR-192. In an additional embodiment, the miR geneproduct is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25,miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183,miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b,miR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2,miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, ormiR-175. In a further embodiment, the miR gene product is not miR-21,miR-301, miR-142 as, miR-142s, miR-194, miR-215, or miR-32. In anotherembodiment, the miR gene product is not miR-148, miR-10a, miR-196-1,miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202,miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the miRgene product is not miR-30, miR-15b, miR-16-2, miR-217, miR-205,miR-204, miR-103, miR-107, miR-9, and miR-137. In a further embodiment,the miR gene product is not miR-145, miR-21, miR-155, miR-10b,miR-125b-1, miR-125b-2, let7a-2, let7a-3, let-7d, miR-122a, miR-191,miR-206, miR-210, let-71, miR-009-1 (miR131-1), miR-34 (miR-170),miR-102 (miR-29b), miR-123 (miR-126), miR-140-as, miR-125a, miR-194,miR-204, miR-213, let-7f-2, miR-101, miR-128b, miR-136, miR-143,miR-149, miR-191, miR-196-1, miR-196-2, miR-202, miR-103-1, or miR-30c.In another embodiment, the miR gene product is not miR-21, miR-125b-1,let-7a-2, let-71, miR-100, let-7g, miR-31, miR-32a-1, miR-33b,miR-34a-2, miR-101-1, miR-135-1, miR-142 as, miR-142s, miR-144, miR-301,miR-29c, miR-30c, miR-106a, or miR-29b-1.

For example, when a miR gene product is down-regulated in a cancer cellin a subject, administering an effective amount of an isolated miR geneproduct to the subject can inhibit proliferation of the cancer cell. Theisolated miR gene product that is administered to the subject can beidentical to the endogenous wild-type miR gene product (e.g., a miR geneproduct shown in Table 1a or Table 1b) that is down-regulated in thecancer cell or it can be a variant or biologically-active fragmentthereof. As defined herein, a “variant” of a miR gene product refers toa miRNA that has less than 100% identity to a corresponding wild-typemiR gene product and possesses one or more biological activities of thecorresponding wild-type miR gene product. Examples of such biologicalactivities include, but are not limited to, inhibition of expression ofa target RNA molecule (e.g., inhibiting translation of a target RNAmolecule, modulating the stability of a target RNA molecule, inhibitingprocessing of a target RNA molecule) and inhibition of a cellularprocess associated with solid cancer (e.g., cell differentiation, cellgrowth, cell death). These variants include species variants andvariants that are the consequence of one or more mutations (e.g., asubstitution, a deletion, an insertion) in a miR gene. In certainembodiments, the variant is at least about 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99% identical to a corresponding wild-type miR gene product.

As defined herein, a “biologically-active fragment” of a miR geneproduct refers to an RNA fragment of a miR gene product that possessesone or more biological activities of a corresponding wild-type miR geneproduct. As described above, examples of such biological activitiesinclude, but are not limited to, inhibition of expression of a targetRNA molecule and inhibition of a cellular process associated with solidcancer. In certain embodiments, the biologically-active fragment is atleast about 5, 7, 10, 12, 15, or 17 nucleotides in length. In aparticular embodiment, an isolated miR gene product can be administeredto a subject in combination with one or more additional anti-cancertreatments. Suitable anti-cancer treatments include, but are not limitedto, chemotherapy, radiation therapy and combinations thereof (e.g.,chemoradiation).

When the at least one isolated miR gene product is up-regulated in thecancer cells, the method comprises administering to the subject aneffective amount of at least one compound for inhibiting expression ofthe at least one miR gene product, referred to herein as miR geneexpression-inhibition compounds, such that proliferation of solid cancercells is inhibited. In a particular embodiment, the at least one miRexpression-inhibition compound is specific for a miR gene productselected from the group consisting of miR-21, miR-17-5p, miR-191,miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146,miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214,miR-30c, miR-25, miR-221, miR-106a and combinations thereof. A miR geneexpression-inhibiting compound can be administered to a subject incombination with one or more additional anti-cancer treatments. Suitableanti-cancer treatments include, but are not limited to, chemotherapy,radiation therapy and combinations thereof (e.g., chemoradiation).

The terms “treat”, “treating” and “treatment”, as used herein, refer toameliorating symptoms associated with a disease or condition, forexample, a solid cancer, including preventing or delaying the onset ofthe disease symptoms, and/or lessening the severity or frequency ofsymptoms of the disease or condition. The terms “subject”, “patient” and“individual” are defined herein to include animals, such as mammals,including, but not limited to, primates, cows, sheep, goats, horses,dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine,equine, canine, feline, rodent, or murine species. In a preferredembodiment, the animal is a human.

As used herein, an “effective amount” of an isolated miR gene product isan amount sufficient to inhibit proliferation of a cancer cell in asubject suffering from a solid cancer. One skilled in the art canreadily determine an effective amount of a miR gene product to beadministered to a given subject, by taking into account factors, such asthe size and weight of the subject; the extent of disease penetration;the age, health and sex of the subject; the route of administration; andwhether the administration is regional or systemic.

For example, an effective amount of an isolated miR gene product can bebased on the approximate weight of a tumor mass to be treated. Theapproximate weight of a tumor mass can be determined by calculating theapproximate volume of the mass, wherein one cubic centimeter of volumeis roughly equivalent to one gram. An effective amount of the isolatedmiR gene product based on the weight of a tumor mass can be in the rangeof about 10-500 micrograms/gram of tumor mass. In certain embodiments,the tumor mass can be at least about 10 micrograms/gram of tumor mass,at least about 60 micrograms/gram of tumor mass or at least about 100micrograms/gram of tumor mass.

An effective amount of an isolated miR gene product can also be based onthe approximate or estimated body weight of a subject to be treated.Preferably, such effective amounts are administered parenterally orenterally, as described herein. For example, an effective amount of theisolated miR gene product is administered to a subject can range fromabout 5 3000 micrograms/kg of body weight, from about 700-1000micrograms/kg of body weight, or greater than about 1000 micrograms/kgof body weight.

One skilled in the art can also readily determine an appropriate dosageregimen for the administration of an isolated miR gene product to agiven subject. For example, a miR gene product can be administered tothe subject once (e.g., as a single injection or deposition).Alternatively, a miR gene product can be administered once or twicedaily to a subject for a period of from about three to abouttwenty-eight days, more particularly from about seven to about ten days.In a particular dosage regimen, a miR gene product is administered oncea day for seven days. Where a dosage regimen comprises multipleadministrations, it is understood that the effective amount of the miRgene product administered to the subject can comprise the total amountof gene product administered over the entire dosage regimen.

As used herein, an “isolated” miR gene product is one that issynthesized, or altered or removed from the natural state through humanintervention. For example, a synthetic miR gene product, or a miR geneproduct partially or completely separated from the coexisting materialsof its natural state, is considered to be “isolated.” An isolated miRgene product can exist in substantially-purified form, or can exist in acell into which the miR gene product has been delivered. Thus, a miRgene product that is deliberately delivered to, or expressed in, a cellis considered an “isolated” miR gene product. A miR gene productproduced inside a cell from a miR precursor molecule is also consideredto be an “isolated” molecule. According to the invention, the isolatedmiR gene products described herein can be used for the manufacture of amedicament for treating a solid cancer in a subject (e.g., a human).

Isolated miR gene products can be obtained using a number of standardtechniques. For example, the miR gene products can be chemicallysynthesized or recombinantly produced using methods known in the art. Inone embodiment, miR gene products are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include, e.g., Proligo (Hamburg,Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical(part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research(Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem(Glasgow, UK).

Alternatively, the miR gene products can be expressed from recombinantcircular or linear DNA plasmids using any suitable promoter. Suitablepromoters for expressing RNA from a plasmid include, e.g., the U6 or H1RNA pol III promoter sequences, or the cytomegalovirus promoters.Selection of other suitable promoters is within the skill in the art.The recombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the miR gene products in cancercells.

The miR gene products that are expressed from recombinant plasmids canbe isolated from cultured cell expression systems by standardtechniques. The miR gene products that are expressed from recombinantplasmids can also be delivered to, and expressed directly in, the cancercells. The use of recombinant plasmids to deliver the miR gene productsto cancer cells is discussed in more detail below.

The miR gene products can be expressed from a separate recombinantplasmid, or they can be expressed from the same recombinant plasmid. Inone embodiment, the miR gene products are expressed as RNA precursormolecules from a single plasmid, and the precursor molecules areprocessed into the functional miR gene product by a suitable processingsystem, including, but not limited to, processing systems extant withina cancer cell. Other suitable processing systems include, e.g., the invitro Drosophila cell lysate system (e.g., as described in U.S.Published Patent Application No. 2002/0086356 to Tuschl et al., theentire disclosure of which is incorporated herein by reference) and theE. coli RNAse III system (e.g., as described in U.S. Published PatentApplication No. 2004/0014113 to Yang et al., the entire disclosure ofwhich is incorporated herein by reference).

Selection of plasmids suitable for expressing the miR gene products,methods for inserting nucleic acid sequences into the plasmid to expressthe gene products, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for example,Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat.Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553;Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al.(2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol.20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, theentire disclosures of which are incorporated herein by reference.

In one embodiment, a plasmid expressing the miR gene products comprisesa sequence encoding a miR precursor RNA under the control of the CMVintermediate-early promoter. As used herein, “under the control” of apromoter means that the nucleic acid sequences encoding the miR geneproduct are located 3′ of the promoter, so that the promoter caninitiate transcription of the miR gene product coding sequences.

The miR gene products can also be expressed from recombinant viralvectors. It is contemplated that the miR gene products can be expressedfrom two separate recombinant viral vectors, or from the same viralvector. The RNA expressed from the recombinant viral vectors can eitherbe isolated from cultured cell expression systems by standardtechniques, or can be expressed directly in cancer cells. The use ofrecombinant viral vectors to deliver the miR gene products to cancercells is discussed in more detail below.

The recombinant viral vectors of the invention comprise sequencesencoding the miR gene products and any suitable promoter for expressingthe RNA sequences. Suitable promoters include, but are not limited to,the U6 or H1 RNA pol III promoter sequences, or the cytomegaloviruspromoters. Selection of other suitable promoters is within the skill inthe art. The recombinant viral vectors of the invention can alsocomprise inducible or regulatable promoters for expression of the miRgene products in a cancer cell.

Any viral vector capable of accepting the coding sequences for the miRgene products can be used; for example, vectors derived from adenovirus(AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses(LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.The tropism of the viral vectors can be modified by pseudotyping thevectors with envelope proteins or other surface antigens from otherviruses, or by substituting different viral capsid proteins, asappropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorsthat express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz, J. E., et al. (2002), J. Virol.76:791-801, the entire disclosure of which is incorporated herein byreference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingRNA into the vector, methods of delivering the viral vector to the cellsof interest, and recovery of the expressed RNA products are within theskill in the art. See, for example, Dornburg (1995), Gene Therapy2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum.Gene Therapy 1:5-14; and Anderson (1998), Nature 392:25-30, the entiredisclosures of which are incorporated herein by reference.

Particularly suitable viral vectors are those derived from AV and AAV. Asuitable AV vector for expressing the miR gene products, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia et al. (2002), Nat.Biotech. 20:1006-1010, the entire disclosure of which is incorporatedherein by reference. Suitable AAV vectors for expressing the miR geneproducts, methods for constructing the recombinant AAV vector, andmethods for delivering the vectors into target cells are described inSamulski et al. (1987), J. Virol. 61:3096-3101; Fisher et al. (1996), J.Virol., 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are incorporated herein byreference. In one embodiment, the miR gene products are expressed from asingle recombinant AAV vector comprising the CMV intermediate earlypromoter.

In a certain embodiment, a recombinant AAV viral vector of the inventioncomprises a nucleic acid sequence encoding a miR precursor RNA inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter. As used herein, “in operable connection witha polyT termination sequence” means that the nucleic acid sequencesencoding the sense or antisense strands are immediately adjacent to thepolyT termination signal in the 5′ direction. During transcription ofthe miR sequences from the vector, the polyT termination signals act toterminate transcription.

In other embodiments of the treatment methods of the invention, aneffective amount of at least one compound that inhibits miR expressioncan be administered to the subject. As used herein, “inhibiting miRexpression” means that the production of the precursor and/or active,mature form of miR gene product after treatment is less than the amountproduced prior to treatment. One skilled in the art can readilydetermine whether miR expression has been inhibited in a cancer cell,using, for example, the techniques for determining miR transcript leveldiscussed above for the diagnostic method. Inhibition can occur at thelevel of gene expression (i.e., by inhibiting transcription of a miRgene encoding the miR gene product) or at the level of processing (e.g.,by inhibiting processing of a miR precursor into a mature, active miR).

As used herein, an “effective amount” of a compound that inhibits miRexpression is an amount sufficient to inhibit proliferation of a cancercell in a subject suffering from a cancer (e.g., a solid cancer). Oneskilled in the art can readily determine an effective amount of a miRexpression-inhibition compound to be administered to a given subject, bytaking into account factors, such as the size and weight of the subject;the extent of disease penetration; the age, health and sex of thesubject; the route of administration; and whether the administration isregional or systemic.

For example, an effective amount of the expression-inhibition compoundcan be based on the approximate weight of a tumor mass to be treated, asdescribed herein. An effective amount of a compound that inhibits miRexpression can also be based on the approximate or estimated body weightof a subject to be treated, as described herein.

One skilled in the art can also readily determine an appropriate dosageregimen for administering a compound that inhibits miR expression to agiven subject.

Suitable compounds for inhibiting miR gene expression includedouble-stranded RNA (such as short- or small-interfering RNA or“siRNA”), antisense nucleic acids, and enzymatic RNA molecules, such asribozymes. Each of these compounds can be targeted to a given miR geneproduct and interfere with the expression of (e.g., inhibit translationof, induce cleavage or destruction of) the target miR gene product.

For example, expression of a given miR gene can be inhibited by inducingRNA interference of the miR gene with an isolated double-stranded RNA(“dsRNA”) molecule which has at least 90%, for example at least 95%, atleast 98%, at least 99%, or 100%, sequence homology with at least aportion of the miR gene product. In a particular embodiment, the dsRNAmolecule is a “short or small interfering RNA” or “siRNA.”

siRNA useful in the present methods comprise short double-stranded RNAfrom about 17 nucleotides to about 29 nucleotides in length, preferablyfrom about 19 to about 25 nucleotides in length. The siRNA comprise asense RNA strand and a complementary antisense RNA strand annealedtogether by standard Watson-Crick base-pairing interactions (hereinafter“base-paired”). The sense strand comprises a nucleic acid sequence thatis substantially identical to a nucleic acid sequence contained withinthe target miR gene product.

As used herein, a nucleic acid sequence in an siRNA which is“substantially identical” to a target sequence contained within thetarget mRNA is a nucleic acid sequence that is identical to the targetsequence, or that differs from the target sequence by one or twonucleotides. The sense and antisense strands of the siRNA can comprisetwo complementary, single-stranded RNA molecules, or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area.

The siRNA can also be altered RNA that differs from naturally-occurringRNA by the addition, deletion, substitution and/or alteration of one ormore nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siRNA or to one ormore internal nucleotides of the siRNA, or modifications that make thesiRNA resistant to nuclease digestion, or the substitution of one ormore nucleotides in the siRNA with deoxyribonucleotides.

One or both strands of the siRNA can also comprise a 3′ overhang. Asused herein, a “3′ overhang” refers to at least one unpaired nucleotideextending from the 3′-end of a duplexed RNA strand. Thus, in certainembodiments, the siRNA comprises at least one 3′ overhang of from 1 toabout 6 nucleotides (which includes ribonucleotides ordeoxyribonucleotides) in length, from 1 to about 5 nucleotides inlength, from 1 to about 4 nucleotides in length, or from about 2 toabout 4 nucleotides in length. In a particular embodiment, the 3′overhang is present on both strands of the siRNA, and is 2 nucleotidesin length. For example, each strand of the siRNA can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

The siRNA can be produced chemically or biologically, or can beexpressed from a recombinant plasmid or viral vector, as described abovefor the isolated miR gene products. Exemplary methods for producing andtesting dsRNA or siRNA molecules are described in U.S. Published PatentApplication No. 2002/0173478 to Gewirtz and in U.S. Published PatentApplication No. 2004/0018176 to Reich et al., the entire disclosures ofboth of which are incorporated herein by reference.

Expression of a given miR gene can also be inhibited by an antisensenucleic acid. As used herein, an “antisense nucleic acid” refers to anucleic acid molecule that binds to target RNA by means of RNA-RNA,RNA-DNA or RNA-peptide nucleic acid interactions, which alters theactivity of the target RNA. Antisense nucleic acids suitable for use inthe present methods are single-stranded nucleic acids (e.g., RNA, DNA,RNA-DNA chimeras, peptide nucleic acid (PNA)) that generally comprise anucleic acid sequence complementary to a contiguous nucleic acidsequence in a miR gene product. The antisense nucleic acid can comprisea nucleic acid sequence that is 50-100% complementary, 75-100%complementary, or 95-100% complementary to a contiguous nucleic acidsequence in a miR gene product. Nucleic acid sequences for the miR geneproducts are provided in Tables 1a and 1b. Without wishing to be boundby any theory, it is believed that the antisense nucleic acids activateRNase H or another cellular nuclease that digests the miR geneproduct/antisense nucleic acid duplex.

Antisense nucleic acids can also contain modifications to the nucleicacid backbone or to the sugar and base moieties (or their equivalent) toenhance target specificity, nuclease resistance, delivery or otherproperties related to efficacy of the molecule. Such modificationsinclude cholesterol moieties, duplex intercalators, such as acridine, orone or more nuclease-resistant groups.

Antisense nucleic acids can be produced chemically or biologically, orcan be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing are within the skill in the art; see, e.g.,Stein and Cheng (1993), Science 261:1004 and U.S. Pat. No. 5,849,902 toWoolf et al., the entire disclosures of which are incorporated herein byreference.

Expression of a given miR gene can also be inhibited by an enzymaticnucleic acid. As used herein, an “enzymatic nucleic acid” refers to anucleic acid comprising a substrate binding region that hascomplementarity to a contiguous nucleic acid sequence of a miR geneproduct, and which is able to specifically cleave the miR gene product.The enzymatic nucleic acid substrate binding region can be, for example,50-100% complementary, 75-100% complementary, or 95-100% complementaryto a contiguous nucleic acid sequence in a miR gene product. Theenzymatic nucleic acids can also comprise modifications at the base,sugar, and/or phosphate groups. An exemplary enzymatic nucleic acid foruse in the present methods is a ribozyme.

The enzymatic nucleic acids can be produced chemically or biologically,or can be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing dsRNA or siRNA molecules are described inWerner and Uhlenbeck (1995), Nucl. Acids Res. 23:2092-96; Hammann et al.(1999), Antisense and Nucleic Acid Drug Dev. 9:25-31; and U.S. Pat. No.4,987,071 to Cech et al, the entire disclosures of which areincorporated herein by reference.

Administration of at least one miR gene product, or at least onecompound for inhibiting miR expression, will inhibit the proliferationof cancer cells in a subject who has a solid cancer. As used herein, to“inhibit the proliferation of a cancer cell” means to kill the cell, orpermanently or temporarily arrest or slow the growth of the cell.Inhibition of cancer cell proliferation can be inferred if the number ofsuch cells in the subject remains constant or decreases afteradministration of the miR gene products or miR geneexpression-inhibition compounds. An inhibition of cancer cellproliferation can also be inferred if the absolute number of such cellsincreases, but the rate of tumor growth decreases.

The number of cancer cells in the body of a subject can be determined bydirect measurement, or by estimation from the size of primary ormetastatic tumor masses. For example, the number of cancer cells in asubject can be measured by immunohistological methods, flow cytometry,or other techniques designed to detect characteristic surface markers ofcancer cells.

The size of a tumor mass can be ascertained by direct visualobservation, or by diagnostic imaging methods, such as X-ray, magneticresonance imaging, ultrasound, and scintigraphy. Diagnostic imagingmethods used to ascertain size of the tumor mass can be employed with orwithout contrast agents, as is known in the art. The size of a tumormass can also be ascertained by physical means, such as palpation of thetissue mass or measurement of the tissue mass with a measuringinstrument, such as a caliper.

The miR gene products or miR gene expression-inhibition compounds can beadministered to a subject by any means suitable for delivering thesecompounds to cancer cells of the subject. For example, the miR geneproducts or miR expression-inhibition compounds can be administered bymethods suitable to transfect cells of the subject with these compounds,or with nucleic acids comprising sequences encoding these compounds. Inone embodiment, the cells are transfected with a plasmid or viral vectorcomprising sequences encoding at least one miR gene product or miR geneexpression-inhibition compound.

Transfection methods for eukaryotic cells are well known in the art, andinclude, e.g., direct injection of the nucleic acid into the nucleus orpronucleus of a cell; electroporation; liposome transfer or transfermediated by lipophilic materials; receptor-mediated nucleic aciddelivery, bioballistic or particle acceleration; calcium phosphateprecipitation, and transfection mediated by viral vectors.

For example, cells can be transfected with a liposomal transfercompound, e.g., DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate,Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN. The amount ofnucleic acid used is not critical to the practice of the invention;acceptable results may be achieved with 0.1-100 micrograms of nucleicacid/10⁵ cells. For example, a ratio of about 0.5 micrograms of plasmidvector in 3 micrograms of DOTAP per 10⁵ cells can be used.

A miR gene product or miR gene expression-inhibition compound can alsobe administered to a subject by any suitable enteral or parenteraladministration route. Suitable enteral administration routes for thepresent methods include, e.g., oral, rectal, or intranasal delivery.Suitable parenteral administration routes include, e.g., intravascularadministration (e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g., peri-tumoral and intra-tumoral injection, intra-retinalinjection, or subretinal injection); subcutaneous injection ordeposition, including subcutaneous infusion (such as by osmotic pumps);direct application to the tissue of interest, for example by a catheteror other placement device (e.g., a retinal pellet or a suppository or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Particularly suitable administration routes are injection,infusion and direct injection into the tumor.

In the present methods, a miR gene product or miR gene productexpression-inhibition compound can be administered to the subject eitheras naked RNA, in combination with a delivery reagent, or as a nucleicacid (e.g., a recombinant plasmid or viral vector) comprising sequencesthat express the miR gene product or miR gene productexpression-inhibition compound. Suitable delivery reagents include,e.g., the Mirus Transit TKO lipophilic reagent; lipofectin;lipofectamine; cellfectin; polycations (e.g., polylysine), andliposomes.

Recombinant plasmids and viral vectors comprising sequences that expressthe miR gene products or miR gene expression-inhibition compounds, andtechniques for delivering such plasmids and vectors to cancer cells, arediscussed herein and/or are well known in the art.

In a particular embodiment, liposomes are used to deliver a miR geneproduct or miR gene expression-inhibition compound (or nucleic acidscomprising sequences encoding them) to a subject. Liposomes can alsoincrease the blood half-life of the gene products or nucleic acids.Suitable liposomes for use in the invention can be formed from standardvesicle-forming lipids, which generally include neutral or negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of factors, such as thedesired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng.9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369, the entire disclosures of which are incorporated herein byreference.

The liposomes for use in the present methods can comprise a ligandmolecule that targets the liposome to cancer cells. Ligands that bind toreceptors prevalent in cancer cells, such as monoclonal antibodies thatbind to tumor cell antigens, are preferred.

The liposomes for use in the present methods can also be modified so asto avoid clearance by the mononuclear macrophage system (“MMS”) andreticuloendothelial system (“RES”). Such modified liposomes haveopsonization-inhibition moieties on the surface or incorporated into theliposome structure. In a particularly preferred embodiment, a liposomeof the invention can comprise both an opsonization-inhibition moiety anda ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization-inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is incorporated herein byreference.

Opsonization-inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate; synthetic polymers, such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcoholand polyxylitol to which carboxylic or amino groups are chemicallylinked, as well as gangliosides, such as ganglioside GM1. Copolymers ofPEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are alsosuitable. In addition, the opsonization-inhibiting polymer can be ablock copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. Theopsonization-inhibiting polymers can also be natural polysaccharidescontaining amino acids or carboxylic acids, e.g., galacturonic acid,glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid,neuraminic acid, alginic acid, carrageenan; aminated polysaccharides oroligosaccharides (linear or branched); or carboxylated polysaccharidesor oligosaccharides, e.g., reacted with derivatives of carbonic acidswith resultant linking of carboxylic groups. Preferably, theopsonization-inhibiting moiety is a PEG, PPG, or a derivative thereof.Liposomes modified with PEG or PEG-derivatives are sometimes called“PEGylated liposomes.”

The opsonization-inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects, for example, solid tumors, will efficiently accumulate theseliposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., U.S.A.,18:6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation ofthe liposomes in the liver and spleen. Thus, liposomes that are modifiedwith opsonization-inhibition moieties are particularly suited to deliverthe miR gene products or miR gene expression-inhibition compounds (ornucleic acids comprising sequences encoding them) to tumor cells.

The miR gene products or miR gene expression-inhibition compounds can beformulated as pharmaceutical compositions, sometimes called“medicaments,” prior to administering them to a subject, according totechniques known in the art. Accordingly, the invention encompassespharmaceutical compositions for treating a solid cancer. In oneembodiment, the pharmaceutical composition comprises at least oneisolated miR gene product, or an isolated variant or biologically-activefragment thereof, and a pharmaceutically-acceptable carrier. In aparticular embodiment, the at least one miR gene product corresponds toa miR gene product that has a decreased level of expression in solidcancer cells relative to suitable control cells. In certain embodimentsthe isolated miR gene product is selected from the group consisting ofmiR-145, miR-155, miR-218-2 combinations thereof.

In other embodiments, the pharmaceutical compositions of the inventioncomprise at least one miR expression-inhibition compound. In aparticular embodiment, the at least one miR gene expression-inhibitioncompound is specific for a miR gene whose expression is greater in solidcancer cells than control cells. In certain embodiments, the miR geneexpression-inhibition compound is specific for one or more miR geneproducts selected from the group consisting of miR-21, miR-17-5p,miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2,miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2,miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof.

Pharmaceutical compositions of the present invention are characterizedas being at least sterile and pyrogen-free. As used herein,“pharmaceutical compositions” include formulations for human andveterinary use. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art, for example as described inRemington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), the entire disclosure of which is incorporatedherein by reference.

The present pharmaceutical compositions comprise at least one miR geneproduct or miR gene expression-inhibition compound (or at least onenucleic acid comprising sequences encoding them) (e.g., 0.1 to 90% byweight), or a physiologically-acceptable salt thereof, mixed with apharmaceutically-acceptable carrier. In certain embodiments, thepharmaceutical compositions of the invention additionally comprise oneor more anti-cancer agents (e.g., chemotherapeutic agents).Thepharmaceutical formulations of the invention can also comprise at leastone miR gene product or miR gene expression-inhibition compound (or atleast one nucleic acid comprising sequences encoding them), which areencapsulated by liposomes and a pharmaceutically-acceptable carrier. Inone embodiment, the pharmaceutical composition comprises a miR gene orgene product that is not miR-15 and/or miR-16.

Especially suitable pharmaceutically-acceptable carriers are water,buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronicacid and the like.

In a particular embodiment, the pharmaceutical compositions of theinvention comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them) that is resistant to degradation by nucleases.One skilled in the art can readily synthesize nucleic acids that arenuclease resistant, for example, by incorporating one or moreribonucleotides that is modified at the 2′-position into the miR geneproduct. Suitable 2′-modified ribonucleotides include those modified atthe 2′-position with fluoro, amino, alkyl, alkoxy, and O-allyl.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude, e.g., physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (such as, for example,calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calciumor sodium salts (for example, calcium chloride, calcium ascorbate,calcium gluconate or calcium lactate). Pharmaceutical compositions ofthe invention can be packaged for use in liquid form, or can belyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically-acceptable carriers can be used; forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of the at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them). A pharmaceutical composition for aerosol(inhalational) administration can comprise 0.01-20% by weight,preferably 1%-10% by weight, of the at least one miR gene product or miRgene expression-inhibition compound (or at least one nucleic acidcomprising sequences encoding them) encapsulated in a liposome asdescribed above, and a propellant. A carrier can also be included asdesired; e.g., lecithin for intranasal delivery.

The pharmaceutical compositions of the invention can further compriseone or more anti-cancer agents. In a particular embodiment, thecompositions comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them) and at least one chemotherapeutic agent.Chemotherapeutic agents that are suitable for the methods of theinvention include, but are not limited to, DNA-alkylating agents,anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizingagents, tubulin destabilizing agents, hormone antagonist agents,topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors,CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinaseinhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acidsaptamers, and molecularly-modified viral, bacterial and exotoxic agents.Examples of suitable agents for the compositions of the presentinvention include, but are not limited to, cytidine arabinoside,methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin),cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin,methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine,camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide,oxaliplatin, irinotecan, topotecan, leucovorin, carmustine,streptozocin, CPT-11, taxol, tamoxifen, dacarbazine, rituximab,daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, fludarabine,docetaxel, FOLFOX4.

The invention also encompasses methods of identifying an inhibitor oftumorigenesis, comprising providing a test agent to a cell and measuringthe level of at least one miR gene product in the cell. In oneembodiment, the method comprises providing a test agent to a cell andmeasuring the level of at least one miR gene product associated withdecreased expression levels in cancer cells. An increase in the level ofthe miR gene product in the cell after the agent is provided, relativeto a suitable control cell (e.g., agent is not provided), is indicativeof the test agent being an inhibitor of tumorigenesis. In a particularembodiment, at least one miR gene product associated with decreasedexpression levels in cancer cells is selected from the group consistingof miR-145, miR-155, miR-218-2 and combinations thereof.

In other embodiments the method comprises providing a test agent to acell and measuring the level of at least one miR gene product associatedwith increased expression levels in cancer cells. A decrease in thelevel of the miR gene product in the cell after the agent is provided,relative to a suitable control cell (e.g., agent is not provided), isindicative of the test agent being an inhibitor of tumorigenesis. In aparticular embodiment, at least one miR gene product associated withincreased expression levels in cancer cells is selected from the groupconsisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b,miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a,miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a.

Suitable agents include, but are not limited to drugs (e.g., smallmolecules, peptides), and biological macromolecules (e.g., proteins,nucleic acids). The agent can be produced recombinantly, synthetically,or it may be isolated (i.e., purified) from a natural source. Variousmethods for providing such agents to a cell (e.g., transfection) arewell known in the art, and several of such methods are describedhereinabove. Methods for detecting the expression of at least one miRgene product (e.g., Northern blotting, in situ hybridization, RT-PCR,expression profiling) are also well known in the art. Several of thesemethods are also described hereinabove.

The invention will now be illustrated by the following non-limitingexamples.

EXEMPLIFICATION

The following Materials and Methods were used in the Examples:

Samples

A total of 540 samples, including 363 primary tumor samples and 177normal tissues, were used in this study (Table 2). The following solidcancers were represented: lung carcinoma, breast carcinoma, prostatecarcinoma, stomach carcinoma, colon carcinoma and pancreatic endocrinetumors. All samples were obtained with informed consent from eachpatient and were confirmed histologically. Normal samples were pairedwith samples from individuals affected with lung and stomach carcinoma,and from normal individuals for the remaining tissues. All normal breastsamples were obtained by pooling 5 unrelated normal tissues. Total RNAwas isolated from tissues using TRIzol™ reagent (Invitrogen), accordingto manufacturer's instructions. MicroRNA microarrays.

Microarray analysis was performed as previously described (Liu, C.-G.,et al., Proc. Natl. Acad. Sci. USA 101: 11755-11760 (2004)). Briefly, 5μg of total RNA was used for hybridization on miRNA microarray chips.These chips contain gene-specific 40-mer oligonucleotide probes, spottedby contacting technologies and covalently attached to a polymericmatrix. The microarrays were hybridized in 6×SSPE (0.9 M NaCU60 mMNaH₂PO₄.H₂O/8 mM EDTA, pH 7.4)/30% formamide at 25° C. for 18 hr, washedin 0.75×TNT (Tris.HCl/NaCU Tween 20) at 37° C. for 40 min, and processedusing direct detection of the biotin-labeled transcripts bystreptavidin-Alexa647 (Molecular Probes) conjugate. Processed slideswere scanned using a microarray scanner (GenePix Pro, Axon), with thelaser set to 635 nm, at fixed PMT setting and a scan resolution of 10mm. The data were confirmed by Northern blotting as described (Calin, G.A., et al., Proc. Natl. Acad. Sci. USA 101:11755-11760 (2004); Iorio, M.V., et al., Cancer Res. 65: 7065-7070 (2005)).

TABLE 2 Samples used in the study (tumors and corresponding normals).Tumour type Cancer Samples Normal Samples Lung carcinoma 123 123  Breastcarcinoma 79  6* Colon carcinoma 46  8 Gastric carcinoma 20 21 Endocrinepancreatic tumours 39 12 Prostate cancer 56  7 All tissues (527) 363177  *Pools of 5 unrelated normal breast tissues per sample (for a totalof 30 unrelated individuals).

Computational Analysis.

Microarray images were analyzed using GenePix Pro (Axon). Average valuesof the replicate spots of each miRNA were background-subtracted,normalized and subjected to further analysis. Normalization wasperformed by using a per chip median normalization method, using themedian array as a reference. Finally, miRNAs measured as present in atleast the smallest of the two classes in a dataset were selected. Absentcalls were thresholded to 4.5 prior to statistical analysis. This levelis the average minimum intensity level detected in the experiments.MicroRNA nomenclature was according to the Genome Browser(www.genome.ucsc.edu) and the microRNA database at Sanger Center(Griffiths-Jones, S., Nucleic Acids Res 32: D109-11 (2004)); in case ofdiscrepancies we followed the microRNA database.Differentially-expressed microRNAs were identified by using the t testprocedure within significance analysis of microarrays (SAM)(Tusher, V.G., et al., Proc Natl Acad Sci USA 98: 5116-21 (2001). SAM calculates ascore for each gene on the basis of the change in expression relative tothe standard deviation of all measurements. Within SAM, t test was used.The microRNA signatures were determined by applying nearest shrunkencentroids method. This method identifies a subgroup of genes that bestcharacterizes each solid cancer from its respective normal counterpart.The prediction error was calculated by means of 10-fold crossvalidation, and for each cancer, we obtained the miR signature thatresulted in the minimal prediction error. A resampling test wasperformed by random permutation analysis to compute the p-value of theshared signature.

Example 1 Identification of a microRNA Expression Signature in HumanSolid Cancers Statistics

The combined cancers/normal tissue comparison was conducted using areduced number of lung samples (80 cancer and 40 normal samples), inorder to balance the different tissues numerically, yielding a total of404 samples. For statistical analysis, 137 miRs, whose expression valueswere above 256 (threshold value) in at least 50% of the samples, wereretained from the 228 that were measured. A T test was used to identifydifferentially-expressed microRNAs (Table 3). The p-values of the T testwere corrected for multiple testing procedures and to control Type Ierror rates. Adjusted p-values were obtained by performing resamplingwith 500,000 permutations (Jung, S. H., et al. Biostatistics 6: 157-69(2005)). This analysis was performed in order to evaluate the results byusing the same method as Lu and co-workers (Lu, J., et al., Nature 435:834-8 (2005)).

As an alternative to T test, significance analysis of microarrays (SAM)was used to identify differentially-expressed microRNAs. This procedureallows for the control of false detection rate (FDR). The delta waschosen to result in an FDR less than or equal to 0.01. microRNA subsetswhich result in the best tumor classification, i.e., which best predictthe two classes (cancer and normal), were then identified using themethod of the nearest shrunken centroids, as implemented in PAM(prediction analysis of microarray). The prediction error was calculatedby means of 10-fold cross validation. The microRNAs were selectedyielding the minimum misclassification error after cross-validation.

Results

By T-test, 43 differentially-expressed miRs with an adjusted p-valuebelow 0.05 were obtained (Table 3). Twenty six miRs were overexpressedand 17 were under-expressed relative to corresponding normal tissueswhen the six solid cancers are grouped together (breast, colon, lung,pancreas, prostate, stomach). These results indicated that the spectrumof expressed miRNAs in solid cancers is very different from that ofnormal cells (43 out of 137 miRNAs, 31%). Using SAM, 49 miRNAs wereidentified as differentially-expressed, of which 34 were up-regulated(Table 4). Using PAM, 36 over-expressed miRNAs in cancer (indicated bypositive cancer scores) and 21 down-regulated miRs (indicated bynegative cancer scores) were identified as differentially-expressed(Table 5). However, these analyses are not tailored to identifyalterations in miR expression that consistently result intransformation, because miR expression is heavily tissue-specific (He,L., et al. Nature 435: 828-833 (2005); also see FIG. 1 and FIG. 2).

The clustering of miRs based on expression profiles derived from 363solid cancer and 177 normal samples using 228 miRs is shown in FIG. 1.The tree, which shows a very good separation between the differenttissues, was constructed using 137 different miRNAs that were expressedin at least 50% of the samples used in the study.

TABLE 3 Differentially regulated miRs in 6 solid cancer types vs. normaltissues (T test stats.) *. Cancer Normal Test miR ID Mean Mean stat Rawp Adj p miR-21 #47 11.538663 9.648338 7.861136 2.00E−06 2.00E−06 miR-141#137 9.024091 7.905398 6.238014 2.00E−06 2.00E−06 miR-212 #208 13.54065114.33617 −6.57942 2.00E−06 2.00E−06 miR-128a prec #113 12.3258813.522675 −6.76388 2.00E−06 2.00E−06 miR-138-2 #133 11.739557 13.144746−7.01204 2.00E−06 2.00E−06 miR-218-2 #221 11.279787 12.539366 −7.405572.00E−06 2.00E−06 miR-23b #51 14.169748 15.949736 −8.37744 2.00E−062.00E−06 miR-195 #184 10.343991 9.172985 5.763262 2.00E−06 1.00E−05miR-212 prec #209 12.686966 13.661763 −5.83132 4.00E−06 1.00E−05miR-29b-2 #95 11.27556 9.940731 5.660854 2.00E−06 1.40E−05 miR-199a-1#191 10.032008 8.920183 5.528849 2.00E−06 3.00E−05 miR-9-3 #28 11.46192212.570412 −5.43006 2.00E−06 4.60E−05 miR-128a #114 13.024235 13.856624−5.35102 6.00E−06 7.20E−05 let-7a-1 #1 12.616569 13.455246 −5.353462.00E−06 7.20E−05 let-7b #5 13.42636 14.068521 −5.17701 1.00E−050.000146 miR-16-2 #39 10.460707 9.305895 5.048375 4.00E−06 0.000224miR-199a-2 #192 9.714225 8.759237 4.862553 1.00E−05 0.000494 miR-152prec #151 11.388676 12.357529 −4.83716 2.00E−06 0.00053 miR-16-1 #3810.443169 9.338182 4.755258 1.00E−05 0.00071 miR-30d #72 13.98201714.775206 −4.5707 1.20E−05 0.001476 miR-34a #78 10.675566 9.637694.467301 2.60E−05 0.00217 miR-17-5p #41 11.567244 10.281468 4.3418343.80E−05 0.0034 miR-128b #115 10.930395 9.947746 4.304764 3.80E−050.003912 miR-20a #46 11.409852 10.19284 4.304678 3.20E−05 0.003912miR-181b-1 prec #211 9.577504 8.804294 4.285968 4.80E−05 0.004126miR-132 #121 9.599947 8.775966 4.284737 5.60E−05 0.004126 miR-200b #1959.475221 8.527243 4.221511 4.00E−05 0.0052 let-7a-3 #4 10.4360899.511546 4.08952 0.000104 0.008242 miR-138-1 #132 8.299613 9.200253−4.05204 5.60E−05 0.00931 miR-29c #65 11.291005 10.326912 4.0193850.000144 0.010312 miR-29a #62 11.381359 10.461075 4.013697 0.000150.010398 miR-96 #86 11.37218 12.136636 −3.94825 0.000138 0.012962miR-191 #177 13.498207 12.729872 3.817228 0.000158 0.02015 miR-27a #5910.399338 9.548582 3.715048 0.000344 0.028096 let-7g #15 10.81968810.01157 3.653239 0.000426 0.033874 miR-9-1 #24 10.102819 9.2129883.651886 0.000388 0.033874 miR-125a #107 10.960998 10.005312 3.6513560.000452 0.033874 miR-95 #84 9.435733 8.751331 3.59406 0.000478 0.039594miR-155 #157 12.505359 13.231221 −3.58369 0.000614 0.040394 miR-199b#194 9.755066 9.082751 3.55934 0.000588 0.04314 miR-24-2 #54 12.61169611.612557 3.518774 0.00087 0.048278 let-7e #11 12.497795 13.055093−3.51589 0.00054 0.048354 miR-92-1 #81 16.081074 16.592426 −3.504460.000928 0.049828 * Forty-three miRs have an adjusted p-value lower than0.05. Twenty-six miRs are overexpressed and 17 down-regulated in breast,colon, lung, pancreas, prostate, stomach carcinomas.

TABLE 4 Differentially regulated miRs in 6 solid cancer types vs. normaltissues (SAM, significance analysis of microarrays) *. d. p. q. R. miRID value stdev value value fold miR-21 #47 3.156 0.24 0 0 2.593 miR-23b#51 −3.117 0.212 0 0 0.443 miR-138-2 #133 −2.514 0.2 0 0 0.402 miR-218-2#221 −2.383 0.17 0 0 0.384 miR-29b-2 #95 2.246 0.236 0 0 1.868 miR-128aprec #113 −2.235 0.177 0 0 0.368 miR-195 #184 2.085 0.203 0 0 1.695miR-141 #137 2.08 0.179 0 0 2.459 miR-199a-1 #191 1.987 0.201 0 0 1.945miR-9-3 #28 −1.97 0.204 0 0 0.433 miR-16-2 #39 1.966 0.229 0 0 1.788miR-17-5p #41 1.964 0.296 0 0 0.725 miR-20a #46 1.898 0.283 0 0 0.969miR-16-1 #38 1.87 0.232 0 0 1.447 miR-212 prec #209 −1.854 0.167 0 00.509 miR-34a #78 1.756 0.232 0 0 1.219 miR-152 prec #151 −1.734 0.2 0 00.46 miR-199a-2 #192 1.721 0.196 0 0 1.838 miR-128b #115 1.674 0.228 0 01.266 miR-212 #208 −1.659 0.121 0 0 0.627 let-7a-1 #1 −1.628 0.157 0 00.461 miR-200b #195 1.626 0.225 0 0 1.432 miR-128a #114 −1.619 0.156 0 00.511 miR-29c #65 1.611 0.24 0 0 1.225 let-7a-3 #4 1.581 0.226 0 0 1.109miR-29a #62 1.565 0.229 0 0 1.706 miR-24-2 #54 1.555 0.284 0 0 0.831miR-138-1 #132 −1.551 0.222 0 0 0.432 miR-125a #107 1.541 0.262 0 01.164 miR-106a #99 1.514 0.275 0 0 0.952 miR-132 #121 1.496 0.192 0 02.158 miR-30d #72 −1.491 0.174 0 0 0.424 miR-9-1 #24 1.478 0.244 0 00.763 miR-27a #59 1.448 0.229 0 0 1.174 miR-181b-1 prec #211 1.435 0.180 0 1.525 let-7g #15 1.394 0.221 0 0 1.072 miR-96 #86 −1.384 0.194 0 00.519 miR-191 #177 1.372 0.201 0 0 1.165 miR-93-1 #83 1.363 0.266 0 00.775 miR-136 #130 −1.355 0.267 0 0 0.364 miR-205 #201 1.343 0.309 0 01.281 miR-185 #170 1.287 0.222 0.001 0.001 0.609 miR-125b-1 #109 1.2620.283 0.001 0.001 1.215 miR-10a #30 1.252 0.227 0.001 0.001 1.643 miR-95#84 1.247 0.19 0.001 0.001 1.509 miR-199b #194 1.228 0.189 0.001 0.0011.246 miR-10b #32 1.219 0.232 0.002 0.001 1.342 let-7i #10 1.216 0.2030.002 0.001 1.026 miR-210 #205 1.213 0.237 0.002 0.001 1.088 * Thirtyfive miRs are over-expressed and 14 are down-regulated in breast, colon,lung, pancreas, prostate, stomach carcinomas (Delta = 0.9, FDR = 0.001).

TABLE 5 MicroRNAs selected by PAM (prediction analysis of microarray) in6 solid cancer types vs. normal tissues miR ID Solid cancer score Normaltissues score miR-21 #47 0.0801 −0.2643 miR-138-2 #133 −0.055 0.1815miR-218-2 #221 −0.0535 0.1765 miR-23b #51 −0.0516 0.17 miR-128a prec#113 −0.0498 0.1642 miR-29b-2 #95 0.0457 −0.1508 miR-195 #184 0.0404−0.1333 miR-17-5p #41 0.0383 −0.1263 miR-9-3 #28 −0.0357 0.1176 miR-212prec #209 −0.0342 0.1129 miR-20a #46 0.0322 −0.1061 miR-141 #137 0.0322−0.1061 miR-199a-1 #191 0.0319 −0.1053 miR-16-2 #39 0.0315 −0.1037miR-152 prec #151 −0.0283 0.0933 miR-16-1 #38 0.0277 −0.0913 miR-34a #780.0269 −0.0886 miR-212 #208 −0.0265 0.0875 let-7a-1 #1 −0.0264 0.0872miR-128a #114 −0.0259 0.0855 miR-128b #115 0.0254 −0.0839 miR-24-2 #540.0244 −0.0803 miR-29c #65 0.0224 −0.0738 miR-199a-2 #192 0.0223 −0.0736let-7a-3 #4 0.0221 −0.073 miR-191 #177 0.0188 −0.062 miR-125a #1070.0186 −0.0613 miR-30d #72 −0.0185 0.061 miR-29a #62 0.0184 −0.0608miR-106a #99 0.0177 −0.0584 miR-93-1 #83 0.0163 −0.0537 miR-200b #1950.0159 −0.0524 let-7g #15 0.0158 −0.0521 miR-27a #59 0.0157 −0.0518miR-96 #86 −0.0156 0.0514 let-7b #5 −0.0152 0.0501 miR-138-1 #132−0.0151 0.0499 miR-9-1 #24 0.0136 −0.0448 miR-181b-1 prec #211 0.0134−0.0442 miR-155 #157 −0.0128 0.0423 miR-132 #121 0.0127 −0.0418 miR-136#130 −0.0112 0.037 let-7i #10 0.0103 −0.034 miR-210 #205 0.0074 −0.0245miR-205 #201 0.0073 −0.024 *miR-185 #170 0.0071 −0.0234 miR-24-1 #520.007 −0.023 miR-199b #194 0.0064 −0.021 miR-125b-1 #109 0.006 −0.0199miR-206 prec #203 −0.005 0.0166 miR-10a #30 0.0045 −0.015 miR-95 #840.0045 −0.0149 let-7c #11 −0.0039 0.013 miR-124a-3 #106 −0.0028 0.0091miR-10b #32 0.002 −0.0066 miR-185 prec #171 −0.0014 0.0047 miR-92-1 #81−2.00E−04 5.00E−04 *T = 1.5 and misclassification error = 0.176. Thirtysix over-expressed miRs in cancer are indicated by positive cancerscores; 21 down-regulated miRs are indicated by negative cancer scores.

Example 2 Identification of microRNA Expression Signatures Associatedwith Various Human Solid Cancers Results

To identify microRNAs that are prognostic for cancer status associatedwith solid tumors, without incurring bias due to tissue specificity, analternative approach was used. First, six tissue-specific signatures,one for each cancer histotype, were obtained by performing independentPAM tests (summarized in Tables 6 and 7) Specific signatures for eachcancer are shown in Tables 8-13: e.g., breast-Table 8; colon-Table 9;lung-Table 10; pancreas-Table 11; prostate-Table 12; stomach-Table 13.Using these data, deregulated microRNAs that were shared among thedifferent histotype miRNA signatures were identified (Table 14). Inorder to compute the p-values for this comparative analysis, are-sampling test with 1,000,000 random permutations on the miRNAidentity was performed. The p-value was defined as the relativefrequency of simulation scores exceeding the real score. Twenty-onemisregulated microRNAs that were common to at least 3 types of solidcancers (p-value=2.5×10⁻³) were identified (Table 14).

TABLE 6 MicroRNAs used to classify human cancers and normal tissues*.Up- Down- Misclassification regulated regulated error after 10 foldCancer miRs miRs cross validation Breast 15 12 0.08 Colon 21 1 0.09 Lung35 3 0.31 Pancreas 55 2 0.02 Prostate 39 6 0.11 Stomach 22 6 0.19*Median normalization was performed and the method of the nearestshrunken centroids was used to select predictive miRNAs.

TABLE 7 Deregulated microRNAs in solid common cancers*. PAM Up- SAM Up-PAM Down- SAM Down- Cancer regulated regulated regulated regulatedBreast 15  3 (FDR = 0.33) 12 47 Colon 21 42 (FDR <= 0.06) 1 5 Lung 35 38(FDR <= 0.01) 3 3 Pancreas 55 50 (FDR <= 0.01) 2 8 Stomach 22 22 (FDR =0.06) 6 4 Prostate 39 49 (FDR = 0.06) 6 3 *Prediction analysis ofmicroarrays (PAM) identifies those genes which best characterize cancersand normal tissues, whilst significance analysis of microarrays (SAM)identifies all those which have differential expression in the twoclasses. False detection rates (FDR) computed in SAM are indicated inparenthesis.

TABLE 8 MicroRNAs selected by prediction analysis of microarray (PAM) inbreast cancer (cancer vs. normal tissues) *. miR Cancer score Normalscore miR-21 (#47) 0.0331 −0.4364 miR-29b-2 (#95) 0.0263 −0.3467 miR-146(#144) 0.0182 −0.2391 miR-125b-2 (#111) −0.0174 0.2286 miR-125b-1 (#109)−0.0169 0.222 miR-10b (#32) −0.0164 0.2166 miR-145 (#143) −0.0158 0.2076miR-181a (#158) 0.0153 −0.201 miR-140 (#136) −0.0122 0.1613 miR-213(#160) 0.0116 −0.1527 miR-29a prec (#63) 0.0109 −0.1441 miR-181b-1(#210) 0.0098 −0.1284 miR-199b (#194) 0.0089 −0.1172 miR-29b-1 (#64)0.0084 −0.1111 miR-130a (#120) −0.0076 0.1001 miR-155 (#157) 0.0072−0.0951 let-7a-2 (#3) −0.0042 0.0554 miR-205 (#201) −0.004 0.0533miR-29c (#65) 0.0032 −0.0423 miR-224 (#228) −0.003 0.0399 miR-100 (#91)−0.0021 0.0283 miR-31 (#73) 0.0017 −0.022 miR-30c (#70) −7.00E−04  0.009 miR-17-5p (#41) 7.00E−04 −0.0089 miR-210 (#205) 4.00E−04 −0.0057miR-122a (#101) 4.00E−04 −0.005 miR-16-2 (#39) −1.00E−04   0.0013 * 27miRs selected, misclassification error after cross validation of 0.008.Seventeen overexpressed miRs in cancer are indicated by positive cancerscores; 12 down-regulated miRs are indicated by negative cancer scores.

TABLE 9 MicroRNAs selected by prediction analysis of microarray (PAM) incolon (cancer vs. normal tissues)*. miR Cancer score Normal scoremiR-24-1 (#52) 0.0972 −0.5589 miR-29b-2 (#95) 0.0669 −0.3845 miR-20a(#46) 0.0596 −0.3424 miR-10a (#30) 0.0511 −0.2938 miR-32 (#75) 0.0401−0.2306 miR-203 (#197) 0.0391 −0.2251 miR-106a (#99) 0.0364 −0.2094miR-17-5p (#41) 0.0349 −0.2005 miR-30c (#70) 0.0328 −0.1888 miR-223(#227) 0.0302 −0.1736 miR-126* (#102) 0.0199 −0.1144 miR-128b (#115)0.0177 −0.102 miR-21 (#47) 0.0162 −0.0929 miR-24-2 (#54) 0.0145 −0.0835miR-99b prec (#88) 0.0125 −0.0721 miR-155 (#157) 0.0092 −0.0528 miR-213(#160) 0.0091 −0.0522 miR-150 (#148) 0.0042 −0.0243 miR-107 (#100) 0.003−0.0173 miR-191 (#177) 0.0028 −0.0159 miR-221 (#224) 0.002 −0.0116miR-9-3 (#28) −0.0014 0.0083 *22 miRs selected, misclassification errorafter cross validation of 0.09. Twenty-one over-expressed miRs in cancerare indicated by positive cancer scores; 1 down-regulated miR isindicated by a negative cancer score.

TABLE 10 MicroRNAs selected by prediction analysis of microarray (PAM)in lung cancer (cancer vs. normal tissues)*. miR Cancer score Normalscore miR-21 (#47) 0.175 −0.175 miR-205 (#201) 0.1317 −0.1317 miR-200b(#195) 0.1127 −0.1127 miR-9-1 (#24) 0.1014 −0.1014 miR-210 (#205) 0.0994−0.0994 miR-148 (#146) 0.0737 −0.0737 miR-141 (#137) 0.0631 −0.0631miR-132 (#121) 0.0586 −0.0586 miR-215 (#213) 0.0575 −0.0575 miR-128b(#115) 0.0559 −0.0559 let-7g (#15) 0.0557 −0.0557 miR-16-2 (#39) 0.0547−0.0547 miR-129-1/2 prec (#118) 0.0515 −0.0515 miR-126* (#102) −0.04060.0406 miR-142-as (#139) 0.0366 −0.0366 miR-30d (#72) −0.0313 0.0313miR-30a-5p (#66) −0.0297 0.0297 miR-7-2 (#21) 0.0273 −0.0273 miR-199a-1(#191) 0.0256 −0.0256 miR-127 (#112) 0.0254 −0.0254 miR-34a prec (#79)0.0214 −0.0214 miR-34a (#78) 0.0188 −0.0188 miR-136 (#130) 0.0174−0.0174 miR-202 (#196) 0.0165 −0.0165 miR-196-2 (#188) 0.0134 −0.0134miR-199a-2 (#192) 0.0126 −0.0126 let-7a-2 (#3) 0.0109 −0.0109 miR-124a-1(#104) 0.0081 −0.0081 miR-149 (#147) 0.0079 −0.0079 miR-17-5p (#41)0.0061 −0.0061 miR-196-1 prec (#186) 0.0053 −0.0053 miR-10a (#30) 0.0049−0.0049 miR-99b prec (#88) 0.0045 −0.0045 miR-196-1 (#185) 0.0044−0.0044 miR-199b (#194) 0.0039 −0.0039 miR-191 (#177) 0.0032 −0.0032miR-195 (#184) 7.00E−04 −7.00E−04 miR-155 (#157) 7.00E−04 −7.00E−04 *38miRs selected, misclassification error after cross validation of 0.31.Thirty-five over-expressed miRs in cancer are indicated by positivecancer scores; 3 down-regulated miRs are indicated by negative cancerscores.

TABLE 11 MicroRNAs selected by prediction analysis of microarray (PAM)in pancreatic cancer (cancer vs. normal tissues)*. miR Cancer scoreNormal score miR-103-2 (#96) 0.4746 −1.582 miR-103-1 (#97) 0.4089−1.3631 miR-24-2 (#54) 0.4059 −1.3529 miR-107 (#100) 0.3701 −1.2336miR-100 (#91) 0.3546 −1.182 miR-125b-2 (#111) 0.3147 −1.0489 miR-125b-1(#109) 0.3071 −1.0237 miR-24-1 (#52) 0.2846 −0.9488 miR-191 (#177)0.2661 −0.887 miR-23a (#50) 0.2586 −0.8619 miR-26a-1 (#56) 0.2081−0.6937 miR-125a (#107) 0.1932 −0.644 miR-130a (#120) 0.1891 −0.6303miR-26b (#58) 0.1861 −0.6203 miR-145 (#143) 0.1847 −0.6158 miR-221(#224) 0.177 −0.59 miR-126* (#102) 0.1732 −0.5772 miR-16-2 (#39) 0.1698−0.5659 miR-146 (#144) 0.1656 −0.552 miR-214 (#212) 0.1642 −0.5472miR-99b (#89) 0.1636 −0.5454 miR-128b (#115) 0.1536 −0.512 miR-155(#157) −0.1529 0.5098 miR-29b-2 (#95) 0.1487 −0.4956 miR-29a (#62)0.1454 −0.4848 miR-25 (#55) 0.1432 −0.4775 miR-16-1 (#38) 0.1424 −0.4746miR-99a (#90) 0.1374 −0.4581 miR-224 (#228) 0.1365 −0.4549 miR-30d (#72)0.1301 −0.4336 miR-92-2 (#82) 0.116 −0.3865 miR-199a-1 (#191) 0.1158−0.3861 miR-223 (#227) 0.1141 −0.3803 miR-29c (#65) 0.113 −0.3768miR-30b (#68) 0.1008 −0.3361 miR-129-1/2 (#117) 0.1001 −0.3337 miR-197(#189) 0.0975 −0.325 miR-17-5p (#41) 0.0955 −0.3185 miR-30c (#70) 0.0948−0.316 miR-7-1 (#19) 0.0933 −0.311 miR-93-1 (#83) 0.0918 −0.3061 miR-140(#136) 0.0904 −0.3015 miR-30a-5p (#66) 0.077 −0.2568 miR-132 (#121)0.0654 −0.2179 miR-181b-1 (#210) 0.0576 −0.1918 miR-152 prec (#151)−0.0477 0.1591 miR-23b (#51) 0.0469 −0.1562 miR-20a (#46) 0.0452 −0.1507miR-222 (#225) 0.0416 −0.1385 miR-27a (#59) 0.0405 −0.1351 miR-92-1(#81) 0.0332 −0.1106 miR-21 (#47) 0.0288 −0.0959 miR-129-1/2 prec (#118)0.0282 −0.0939 miR-150 (#148) 0.0173 −0.0578 miR-32 (#75) 0.0167 −0.0558miR-106a (#99) 0.0142 −0.0473 miR-29b-1 (#64) 0.0084 −0.028 *57 miRsselected, misclassification error after cross validation of 0.02.Fifty-seven miRs are over-expressed and 2 are down-regulated in cancer(indicated by positive and negative scores, respectively).

TABLE 12 MicroRNAs selected by prediction analysis of microarray (PAM)in prostate cancer (cancer vs. normal tissues) *. miR Cancer scoreNormal score let-7d (#8) 0.0528 −0.4227 miR-128a prec (#113) −0.04120.3298 miR-195 (#184) 0.04 −0.3199 miR-203 (#197) 0.0356 −0.2851let-7a-2 prec (#2) −0.0313 0.2504 miR-34a (#78) 0.0303 −0.2428 miR-20a(#46) 0.029 −0.2319 miR-218-2 (#221) −0.0252 0.2018 miR-29a (#62) 0.0247−0.1978 miR-25 (#55) 0.0233 −0.1861 miR-95 (#84) 0.0233 −0.1861 miR-197(#189) 0.0198 −0.1587 miR-135-2 (#128) 0.0198 −0.1582 miR-187 (#173)0.0192 −0.1535 miR-196-1 (#185) 0.0176 −0.1411 miR-148 (#146) 0.0175−0.1401 miR-191 (#177) 0.017 −0.136 miR-21 (#47) 0.0169 −0.1351 let-7i(#10) 0.0163 −0.1303 miR-198 (#190) 0.0145 −0.1161 miR-199a-2 (#192)0.0136 −0.1088 miR-30c (#70) 0.0133 −0.1062 miR-17-5p (#41) 0.0132−0.1053 miR-92-2 (#82) 0.012 −0.0961 miR-146 (#144) 0.0113 −0.0908miR-181b-1 prec (#211) 0.011 −0.0878 miR-32 (#75) 0.0109 −0.0873 miR-206(#202) 0.0104 −0.083 miR-184 prec (#169) 0.0096 −0.0764 miR-29a prec(#63) −0.0095 0.076 miR-29b-2 (#95) 0.0092 −0.0739 miR-149 (#147)−0.0084 0.0676 miR-181b-1 (#210) 0.0049 −0.0392 miR-196-1 prec (#186)0.0042 −0.0335 miR-93-1 (#83) 0.0039 −0.0312 miR-223 (#227) 0.0038−0.0308 miR-16-1 (#38) 0.0028 −0.0226 miR-101-1 prec (#92) 0.0015−0.0123 miR-124a-1 (#104) 0.0015 −0.0119 miR-26a-1 (#56) 0.0015 −0.0119miR-214 (#212) 0.0013 −0.0105 miR-27a (#59) 0.0011 −0.0091 miR-24-1(#53) −8.00E−04   0.0067 miR-106a (#99) 7.00E−04 −0.0057 miR-199a-1(#191) 4.00E−04 −0.0029 * T = 1, 45 miRs selected, misclassificationerror after cross validation of 0.11. Thirty-nine over-expressed miRs incancer are indicated by positive cancer scores; 6 downregulated miRs areindicated by negative cancer scores.

TABLE 13 MicroRNAs selected by prediction analysis of microarray (PAM)in stomach cancer (cancer vs. normal tissues) *. miR Cancer score Normalscore miR-223 (#227) 0.1896 −0.1806 miR-21 (#47) 0.1872 −0.1783miR-218-2 (#221) −0.1552 0.1478 miR-103-2 (#96) 0.1206 −0.1148 miR-92-2(#82) 0.1142 −0.1088 miR-25 (#55) 0.1097 −0.1045 miR-136 (#130) −0.10970.1045 miR-191 (#177) 0.0946 −0.0901 miR-221 (#224) 0.0919 −0.0876miR-125b-2 (#111) 0.0913 −0.0869 miR-103-1 (#97) 0.0837 −0.0797 miR-214(#212) 0.0749 −0.0713 miR-222 (#225) 0.0749 −0.0713 miR-212 prec (#209)−0.054 0.0514 miR-125b-1 (#109) 0.0528 −0.0503 miR-100 (#91) 0.0526−0.0501 miR-107 (#100) 0.0388 −0.0369 miR-92-1 (#81) 0.0369 −0.0351miR-96 (#86) −0.0306 0.0291 miR-192 (#178) 0.0236 −0.0224 miR-23a (#50)0.022 −0.021 miR-215 (#213) 0.0204 −0.0194 miR-7-2 (#21) 0.0189 −0.018miR-138-2 (#133) −0.0185 0.0176 miR-24-1 (#52) 0.0151 −0.0144 miR-99b(#89) 0.0098 −0.0093 miR-33b (#76) −0.0049 0.0046 miR-24-2 (#54) 0.0041−0.0039 * T = 1, 28 miRs selected, misclassification error after crossvalidation of 0.19. Twenty-two over-expressed miRs in cancer areindicated by positive cancer scores; 6 down-regulated miRs are indicatedby negative cancer scores.

TABLE 14 The microRNAs shared by the signatures of the 6 solid cancers*.miR N Tumor Type miR-21 6 Breast Colon Lung Pancreas Prostate StomachmiR-17-5p 5 Breast Colon Lung Pancreas Prostate miR-191 5 Colon LungPancreas Prostate Stomach miR-29b-2 4 Breast Colon Pancreas ProstatemiR-223 4 Colon Pancreas Prostate Stomach miR-128b 3 Colon Lung PancreasmiR-199a-1 3 Lung Pancreas Prostate miR-24-1 3 Colon Pancreas StomachmiR-24-2 3 Colon Pancreas Stomach miR-146 3 Breast Pancreas ProstatemiR-155 3 Breast Colon Lung miR-181b-1 3 Breast Pancreas ProstatemiR-20a 3 Colon Pancreas Prostate miR-107 3 Colon Pancreas StomachmiR-32 3 Colon Pancreas Prostate miR-92-2 3 Pancreas Prostate StomachmiR-214 3 Pancreas Prostate Stomach miR-30c 3 Colon Pancreas ProstatemiR-25 3 Pancreas Prostate Stomach miR-221 3 Colon Pancreas StomachmiR-106a 3 Colon Pancreas Prostate *The list includes 21 commonlyup-regulated microRNAs in 3 or more (N) types of solid cancers (p-value= 2.5 × 10⁻³).

To maximize concision, the mean absolute expression levels of thederegulated miRs for the 6 cancer/normal pairs were computed. Using theexpression level of miRs in the comprehensive subset, the differenttissues were correctly classified, irrespective of the disease status(FIG. 3).

FIG. 4 shows differential expression of the common microRNAs across thedifferent tumor tissues, in relation to the normal tissues. The treedisplays the different cancer types according to fold changes in themiRNA subset. Prostate, colon, stomach and pancreatic tissues are mostsimilar among them, while lung and breast tissues were represented by afairly different signature (FIG. 4). This tree clearly shows whichmiRNAs are associated with a particular cancer histotype.

Strikingly, miR-21, miR-191 and miR-17-5p are significantlyover-expressed in all, or in 5 out of 6, of the tumor types that wereconsidered. miR-21 was reported to be over-expressed in glioblastoma andto have anti-apoptotic properties (Chan, J. A., et al., Cancer Res. 65:6029-6033 (2005)). Lung cancer shares a portion of its signature withbreast cancer and a portion with the other solid tumors, includingmiR-17/20/92, all three of which are members of the microRNA clusterthat actively cooperates with c-Myc to accelerate lymphomagenesis (He,L., et al., Nature 435: 828-833 (2005)). The identification of thesemicroRNAs as being over-expressed is an excellent confirmation of ourapproach. A second miRNA group that is activated includes miR-210 andmiR-213, together with miR-155, which was already reported to beamplified in large cell lymphomas (E is, P. S., et al., Proc. Natl.Acad. Sci. USA 102: 3627-3632 (2005)), children with Burkitt lymphoma(Metzler, M., et al., Genes Chromosomes Cancer 39:167-169 (2004)) andvarious B cell lymphomas (Kluiver, J, et al., J. Pathol., e-publishedonline, Jul. 22, 2005). These microRNAs are the only ones up-regulatedin breast and lung cancer. miR-218-2 is consistently down-regulated incolon, stomach, prostate and pancreas cancers, but not in lung andbreast carcinomas.

Several observations strengthen these results. First, in this study, theexpression levels of both the precursor pre-miRNA and the mature miRNAwere determined for the majority of genes. Of note, with the exceptionof miR-212 and miR-128a, in all other instances, theabnormally-expressed region was that corresponding to the active geneproduct. Second, as shown in FIG. 3, the expression variation of themiRNAs in the comprehensive subset was often univocal (namely, down- orup-regulation) across the different types of cancers, suggesting acommon mechanism in human tumorigenesis. Third, the microarray data werevalidated by solution hybridization for 12 breast samples (miR-125b,miR-145 and miR-21; Iorio, M. V., et al., Cancer Res. 65: 7065-7070(2005)) and 17 endocrine pancreatic and normal samples (miR-103, miR-155and miR-204; data not shown), strongly confirming the accuracy of themicroarray data.

Example 3 Identification of Predicted Targets for microRNAs that areDeregulated in Solid Tumors Materials and Methods: Tumor Suppressor andOncogene Target Predictions

The most recent TargetScan predictions (April 2005) were used toidentify putative microRNA targets. These include essentially the 3′UTRtargets reported by Lewis et al. (Lewis, B. P., et al, Cell 120: 15-20(2005)), with a few changes arising from updated gene boundarydefinitions from the April 2005 UCSC Genome Browser mapping of RefSeqmRNAs to the hg17 human genome assembly. Among the putative targets,known cancer genes (tumor suppressors and oncogenes) were specifiedaccording to their identification in the Cancer Gene Census, which isaccessible at the internet site www.sanger.ac.uk/genetics/CGP/Census/,or as reported by OMIM at www.ncbi.nlm.nih.gov.

Target In Vitro Assays

For luciferase reporter experiments, 3′ UTR segments of Rb1, TGFBR2 andPlag1 that are predicted to interact with specific cancer-associatedmicroRNAs were amplified by PCR from human genomic DNA and inserted intothe pGL3 control vector (Promega) using the XbaI site immediatelydownstream from the stop codon of luciferase. The human megakaryocyticcell line, MEG-01, was grown in 10% FBS in RPMI medium 1640,supplemented with 1× nonessential amino acid and 1 mmol sodium pyruvateat 37° C. in a hunified atmosphere of 5% CO₂. The cells wereco-transfected in 12-well plates by using siPORT neoFX (Ambion, Austin,Tex.), according to the manufacturer's protocol, with 0.4 mg of thefirefly luciferase reporter vector and 0.08 μg of the control vectorcontaining Renilla luciferase, pRL-TK (Promega). For each well, microRNAoligonucleotides (Dharmacon Research, Lafayette, Colo.) and anti-senseor scrambled oligonucleotides (Ambion) were used at a concentration of10 nM. Firefly and Renilla luciferase activities were measuredconsecutively at 24 h post transfection using dual-luciferase assays(Promega).

Western Blotting for RB1

Levels of RB1 protein were quantified using a mouse monoclonal anti-RB1antibody (Santa Cruz, Calif.) using standard procedures for Westernblotting. The normalization was performed with mouse monoclonalanti-Actin antibody (Sigma).

Results

The functional significance of microRNA deregulation in cancer needs tobe understood. In solid tumors, it appears that the most common microRNAevent is gain of expression, while loss of expression in cancer is amore limited event, and more tissue specific. We used a three-stepconsequential approach in the following order: first, “in silico”prediction of targets, then luciferase assay for first validation ofcancer relevant targets and finally, ex vivo tumor correlation betweenmiRNA expression (by microarray) and target protein expression (byWestern blotting) for a specific miRNA:mRNA interactor pair. Relevanttargets for cancer miRNAs could be either recessive (e.g., tumorsuppressors) or dominant (e.g., oncogenes) cancer genes. To test thehypothesis that microRNAs that are deregulated in solid tumors targetknown oncogenes or tumor suppressors, the predicted targets for thesemiRNAs were determined using TargetScan, a database of conserved 3′ UTRmicroRNA targets (Lewis, B. P., et al, Cell 120: 15-20 (2005)).TargetScan contained 5,121 predictions for 18 miRNAs that aredysregulated in solid tumors, in the total 22,402 (26.5%) predictions.One hundred fifteen out of 263 (44%) well-known cancer genes werepredicted as targets for these 18 miRNAs (Table 15). Because a highpercentage of cancer genes are targeted by miRs that are deregulated insolid tumors, it is unlikely that these predictions are due to chance(P<0.0001 at Fisher exact-test).

In silico predictions for three different cancer genes, Retinoblastoma(Rb), TGF-beta-2 receptor (TGFBR2), and pleiomorphic adenoma gene 1(PLAG1), were confirmed experimentally by in vitro assays. Using aluciferase reporter assay, three microRNAs tested (miR-106a, miR-20a andmiR-26a-1) caused a significant reduction of protein translationrelative to the scrambled control oligoRNAs in transfected MEG-01 cells(FIG. 6). Retinoblastoma 3′UTR, for example, was found to interactfunctionally with miR-106a. The biological significance of thismiRNA:mRNA interaction is reinforced by previous reports showing thatthe Rb1 gene is normally transcribed in colon cancers, whilst variousfractions of cells do not express Rb1 protein (Ali, A. A., et al., FASEBJ. 7:931-937 (1993)). This finding suggests the existence of apost-transcriptional mechanism for regulating Rb1 that could beexplained by concomitant miR-106a over-expression in colon carcinoma(FIG. 4). Furthermore, mir-20a is down-regulated in breast cancer (FIG.4) and TFGBR2 protein is expressed in the epithelium of breast cancercells (Buck, M. B., et al., Clin. Cancer Res. 10:491-498 (2004)).Conversely, the over-expression of mir-20a in colon cancer may representa novel mechanism for down-regulating TGFBR2, in addition to mutationalinactivation (Biswas, S., et al., Cancer Res. 64:687-692 (2004)).

Finally, a set of patient samples was tested to verify whether RB1protein expression correlates with miR-106a expression (FIG. 5 and FIG.6B). As expected, in gastric, prostate and lung tumor samples RB1 wasdown-regulated (in respect to the paired normal) and miR-106a was foundto be over-expressed, while in breast tumor samples, where miR-106a isslightly down-regulated (FIG. 5 and FIG. 6B), RB1 is expressed atslightly higher levels then in the paired normal control.

These experimental proofs reinforce the hypothesis that key cancer genesare regulated by aberrant expression of miRs in solid cancers. Thesedata add novel examples to the list of microRNA with important cancergene targets, as previously shown by Johnsson et al. (Johnson, S. M., etal., Cell 120: 635-647 (2005)) for the let-7:Ras interaction, O'Donnellet al. (O'Donnell, K. A., et al., Nature 435:839-843 (2005)) for themiR-17-5p:cMyc interaction, and Cimmino et al. (Cimmino, A., et al.,Proc. Natl. Acad. Sci. USA 102:13944-13949 (2005)) for the mir-16:Bcl2interaction. Notably, miR-17-5p and miR-16 are members of the miRNAsolid cancer signature described herein.

TABLE 15 Oncogenes and tumor suppressor genes predicted by TargetScanSas targets or microRNAs from the comprehensive cancer subset.* miRNAgene Gene Name Gene description miR-26a, miR-146 ABL2 v-abl Abelsonmurine leukemia viral oncogene homolog 2 (arg, Abelson-related gene)miR-107 AF5q31 ALL1 fused gene from 5q31 miR-20, miR-125b AKT3 v-aktmurine thymoma viral oncogene homolog 3 miR-26a, miR-155 APCadenomatosis polyposis coli miR-125b miR-26a, miR-218 ARHGEF12 RHOguanine nucleotide exchange factor (GEF) 12 (LARG) miR-107, miR-221 ARNTaryl hydrocarbon receptor nuclear translocator miR-192 ATF1 activatingtranscription factor 1 miR-26a ATM Ataxia telangiectasia mutated(includes complementation groups A, C and D) miR-24 AXL AXL receptortyrosine kinase miR-26a, miR-107, BCL11A B-cell CLL/lymphoma 11AmiR-146, miR-155 miR-138, miR-92 miR-20 BCL11B B-cell CLL/lymphoma 11B(CTIP2) miR-21 BCL2 B-cell CLL/lymphoma 2 miR-26a, miR-26a BCL6 B-cellCLL/lymphoma 6 (zinc finger protein 51) miR-20, miR-92 BCL9 B-cellCLL/lymphoma 9 miR-26a, miR-223 CBFB core-binding factor, beta subunitmiR-221, miR-125b miR-218 CCDC6 coiled-coil domain containing 6 miR-20CCND1 cyclin D1 (PRAD1: parathyroid adenomatosis 1) miR-26a, miR-20CCND2 cyclin D2 miR-26a, miR-107, CDK6 cyclin-dependent kinase 6 miR-92miR-20 CDKN1A cyclin-dependent kinase inhibitor 1A (p21, Cip1) miR-221,miR-92 CDKN1C cyclin-dependent kinase inhibitor 1C (p57, Kip2) miR-24CDX2 caudal type homeo box transcription factor 2 miR-92 CEBPACCAAT/enhancer binding protein (C/EBP), alpha miR-26a CLTC clathrin,heavy polypeptide (Hc) miR-218 COL1A1 collagen, type I, alpha 1 miR-26aCREBBP CREB binding protein (CBP) miR-20 CRK v-crk avian sarcoma virusCT10 oncogene homolog miR-20 CSF1 colony stimulating factor 1(macrophage) miR-221, miR-192 DDX6 DEAD/H (Asp-Glu-Ala-Asp/His) boxpolypeptide 6 (RNA helicase, 54 kD) miR-138 DEK DEK oncogene (DNAbinding) miR-20 E2F1 E2F transcription factor 1 miR-20 ELK3 ELK3,ETS-domain protein (SRF accessory protein 2) miR-24 ELL ELL gene (11-19lysine-rich leukemia gene) miR-26a, miR-138 ERBB4 v-erb-a avianerythroblastic leukemia viral oncogene homolog-like 4 miR-221, miR-155,ETS1 v-ets avian erythroblastosis virus E26 oncogene miR-125b homolog 1miR-20 ETV1 ets variant gene 1 miR-125b ETV6 ets variant gene 6 (TELoncogene) miR-223 FAT FAT tumor suppressor (Drosophila) homolog miR-223,miR-125b, FGFR2 fibroblast growth factor receptor 2 miR-218 miR-92 FLI1Friend leukemia virus integration 1 miR-24, miR-20 FLT1 fms-relatedtyrosine kinase 1 (vascular endothelial growth factor/vascularpermeability factor receptor) miR-221 FOS v-fos FBJ murine osteosarcomaviral oncogene homolog miR-92 FOXG1B forkhead box G1B miR-223 FOXO3Aforkhead box O3A miR-125b GOLGA5 golgi autoantigen, golgin subfamily a,5 (PTC5) miR-138 GPHN gephyrin (GPH) miR-107, miR-223, HLF hepaticleukemia factor miR-20, miR-218 miR-26a, miR-107 HMGA1 high mobilitygroup AT-hook 1 miR-20 HOXA13 homeo box A13 miR-92 HOXA9 homeo box A9miR-125b IRF4 interferon regulatory factor 4 miR-146, miR-20, JAZF1juxtaposed with another zinc finger gene 1 miR-138 miR-92 JUN v-junavian sarcoma virus 17 oncogene homolog miR-155 KRAS v-Ki-ras2 Kirstenrat sarcoma 2 viral oncogene homolog miR-218 LASP1 LIM and SH3 protein 1miR-218 LHFP lipoma HMGIC fusion partner miR-125b, miR-218 LIFR leukemiainhibitory factor receptor miR-223 LMO2 LIM domain only 2(rhombotin-like 1) (RBTN2) miR-223, miR-155, MAF v-mafmusculoaponeurotic fibrosarcoma (avian) miR-125b, miR-92 oncogenehomolog miR-92 MAP2K4 mitogen-activated protein kinase kinase 4 miR-146,miR-20 MAP3K8 mitogen-activated protein kinase kinase kinase 8 miR-125bMAX MAX protein miR-218 MCC mutated in colorectal cancers miR-24 MEN1multiple endocrine neoplasia I miR-138 MLLT6 myeloid/lymphoid ormixed-lineage leukemia (trithorax homolog, Drosophila): translocated to,6 (AF17) miR-192 MSN moesin miR-24 MYB v-myb avian myeloblastosis viraloncogene homolog miR-107, miR-223, MYBL1 v-myb avian myeloblastosisviral oncogene miR-146, miR-221, homolog-like 1 miR-155, miR-218miR-107, miR-20 MYCN v-myc avian myelocytomatosis viral relatedoncogene, neuroblastoma derived miR-107, miR-92 MYH9 myosin, heavypolypeptide 9, non-muscle miR-24 MYST4 MYST histone acetyltransferase(monocytic leukemia) 4 (MORF) miR-20 NBL1 neuroblastoma, suppression oftumorigenicity 1 miR-125b NIN ninein (GSK3B interacting protein)miR-26a, miR-107 NKTR natural killer-tumor recognition sequence miR-92NOTCH1 Notch homolog 1, translocation-associated (Drosophila) (TAN1)miR-24 NTRK3 neurotrophic tyrosine kinase, receptor, type 3 miR-125bPCSK7 proprotein convertase subtilisin/kexin type 7 miR-24, miR-146 PER1period homolog 1 (Drosophila) miR-146, miR-125b, PHOX2B paired-likehomeobox 2b miR-138, miR-155 PICALM phosphatidylinositol bindingclathrin assembly protein (CALM) miR-24, miR-26a PIM1 pim-1 oncogenemiR-24, miR-26a, PLAG1 pleiomorphic adenoma gene 1 miR-21, miR-107,miR-20, miR-155 miR-218 RAB8A RAB8A, member RAS oncogene family miR-24,miR-221 RALA v-ral simian leukemia viral oncogene homolog A (rasrelated) miR-138 RARA retinoic acid receptor, alpha miR-20, miR-192 RB1retinoblastoma 1 (including osteosarcoma) miR-20, RBL1retinoblastoma-like 1 (p107) miR-20 RBL2 retinoblastoma-like 2 (p130)miR-155, miR-138 REL v-rel avian reticuloendotheliosis viral oncogenehomolog miR-20, miR-138 RHOC ras homolog gene family, member C miR-20,miR-192 RUNX1 runt-related transcription factor 1 (AML1) miR-107,miR-223 SEPT6 seplin 6 miR-146, miR-20, SET SET translocation miR-125bmiR-21, miR-20, SKI v-ski avian sarcoma viral oncogene homolog miR-155,miR-218 miR-26a, miR-146 SMAD4 SMAD, mothers against DPP homolog 4(Drosophila) miR-155 SPI1 spleen focus forming virus (SFFV) proviralintegration oncogene spi1 miR-125b SS18 synovial sarcoma translocation,chromosome 18 miR-107, miR-155 SUFU suppressor of fused homolog(Drosophila) miR-92 TAF15 TAF15 RNA polymerase II, TATA box bindingprotein (TBP)-associated factor, 68 kDa miR-26a, miR-221, TCF12transcription factor 12 (HTF4, helix-loop-helix miR-138 transcriptionfactors 4) miR-21, miR-20 TGFBR2 transforming growth factor, betareceptor II (70-80 kD) miR-24, miR-26a, TOP1 topoisomerase (DNA) ImiR-92 miR-138 TPM4 tropomyosin 4 miR-20 TRIP11 thyroid hormone receptorinteractor 11 miR-92 TSC1 Tuberous sclerosis 1 miR-20 TSG101 Tumorsusceptibility gene 101 miR-20 TUSC2 Tumor suppressor candidate 2 miR-24VAV1 vav 1 oncogene miR-125b VAV2 vav 2 oncogene miR-107 WHSC1Wolf-Hirschhorn syndrome candidate 1(MMSET) miR-138 WHSC1L1Wolf-Hirschhorn syndrome candidate 1-like 1 (NSD3) miR-26a WNT5Awingless-type MMTV integration site family, member 5A miR-26a, miR-20,YES1 v-yes-1 Yamaguchi sarcoma viral oncogene miR-125b homolog 1miR-107, miR-221 ZNF198 zinc finger protein 198 miR-218 ZNFN1A1 zincfinger protein, subfamily 1A, 1 (Ikaros) *Known cancer genes (e.g.,tumor suppressors, oncogenes) comprise those identified in the CancerGene Census at www.sanger.ac.uk/genetics/CGP/Census/ or reported by OMIMat www.ncbi.nlm.nih.gov.

The relevant teachings of all publications cited herein that have notexplicitly been incorporated by reference, are incorporated herein byreference in their entirety. While this invention has been particularlyshown and described with references to preferred embodiments thereof, itwill, be understood by those skilled in the art that various changes inform and details may be made therein without departing from the scope ofthe invention encompassed by the appended claims.

1-33. (canceled)
 34. A method of diagnosing whether a subject has, or isat risk for developing, breast cancer, comprising: a.) comparing thelevel of at least miR-146 gene product and at least one additional miRgene product in a test sample from a subject to a control level of atleast one corresponding miR-146 gene product and at least one additionalmiR gene product; and b.) diagnosing whether the subject has, or is atrisk for developing, breast cancer, if the level of the at least onemiR-146 gene product and at least one additional miR gene product in thetest sample from the subject is greater than a control level of thecorresponding miR-146 gene product and at least one additional miR geneproduct.
 35. A method of claim 34, wherein the at least one additionalmiR gene product is miR-21.
 36. A method of claim 34, wherein the atleast one additional miR gene product is miR-29b-2.
 37. A method ofclaim 34, wherein the at least one additional miR gene product ismiR-181a.
 38. A method of claim 34, which further comprises: comparingthe level of at least one additional miR gene product in a test samplefrom the subject to a control level of the miR gene product, wherein theat least one additional miR gene product is selected from the groupconsisting of: miR-21; miR-29b-2; miR-146; miR-125b-2; miR-125b-1;miR-10b; miR-145; miR-181a; miR-140; miR-213; miR-29a prec; miR-181b-1;miR-199b; miR-29b-1; miR-130a; miR-155; let-7a-2; miR-205; miR-29c;miR-224; miR-100; miR-31; miR-30c; miR-17-5p; miR-210; miR-122a; andmiR-16-2.
 39. A method of diagnosing whether a subject has, or is atrisk for developing, breast cancer, comprising: a.) comparing the levelof at least miR-29b-2 gene product and at least one additional miR geneproduct in a test sample from a subject to a control level of at leastone corresponding miR-29b-2 gene product and at least one additional miRgene product; and b.) diagnosing whether the subject has, or is at riskfor developing, breast cancer, if the level of the at least onemiR-29b-2 gene product and at least one additional miR gene product inthe test sample from the subject is greater than a control level of thecorresponding miR-29b-2 gene product and at least one additional miRgene product.
 40. A method of claim 39, wherein the at least oneadditional miR gene product is miR-21.
 41. A method of claim 39, whereinthe at least one additional miR gene product is miR-181a.
 42. A methodof claim 39, which further comprises: comparing the level of at leastone additional miR gene product in a test sample from the subject to acontrol level of the miR gene product, wherein the at least oneadditional miR gene product is selected from the group consisting of:miR-21; miR-29b-2; miR-146; miR-125b-2; miR-125b-1; miR-10b; miR-145;miR-181a; miR-140; miR-213; miR-29a prec; miR-181b-1; miR-199b;miR-29b-1; miR-130a; miR-155; let-7a-2; miR-205; miR-29c; miR-224;miR-100; miR-31; miR-30c; miR-17-5p; miR-210; miR-122a; and miR-16-2.43. A method of diagnosing whether a subject has, or is at risk fordeveloping, breast cancer, comprising: a.) comparing the level of atleast miR-21 gene product and at least one additional miR gene productin a test sample from a subject to a control level of at least onecorresponding miR-21 gene product and at least one additional miR geneproduct; and b.) diagnosing whether the subject has, or is at risk fordeveloping, breast cancer, if the level of the at least one miR-21 geneproduct and at least one additional miR gene product in the test samplefrom the subject is greater than a control level of miR-21 gene productand less than a control level of at least one corresponding additionalmiR gene product.
 44. A method of claim 43, wherein the at least oneadditional miR gene product is miR-125b-2.
 45. A method of claim 43,wherein the at least one additional miR gene product is miR-125b-1. 46.A method of claim 43, wherein the at least one additional miR geneproduct is miR-10b.
 47. A method of claim 43, wherein the at least oneadditional miR gene product is miR-145.
 48. A method of claim 43, whichfurther comprises: comparing the level of at least one additional miRgene product in a test sample from the subject to a control level of themiR gene product, wherein the at least one additional miR gene productis selected from the group consisting of: miR-21; miR-29b-2; miR-146;miR-125b-2; miR-125b-1; miR-10b; miR-145; miR-181a; miR-140; miR-213;miR-29a prec; miR-181b-1; miR-199b; miR-29b-1; miR-130a; miR-155;let-7a-2; miR-205; miR-29c; miR-224; miR-100; miR-31; miR-30c;miR-17-5p; miR-210; miR-122a; and miR-16-2.
 49. A method of diagnosingwhether a subject has, or is at risk for developing, breast cancer,comprising: a.) comparing the level of at least miR-29b-2 gene productand at least one additional miR gene product in a test sample from asubject to a control level of at least one corresponding miR-29b-2 geneproduct and at least one additional miR gene product; and b.) diagnosingwhether the subject has, or is at risk for developing, breast cancer, ifthe level of the at least one miR-29b-2 gene product and at least oneadditional miR gene product in the test sample from the subject isgreater than a control level of miR-29b-2 gene product and less than acontrol level of at least one corresponding additional miR gene product.50. A method of claim 49, wherein the at least one additional miR geneproduct is miR-125b-2.
 51. A method of claim 49, wherein the at leastone additional miR gene product is miR-125b-1.
 52. A method of claim 49,wherein the at least one additional miR gene product is miR-10b.
 53. Amethod of claim 49, wherein the at least one additional miR gene productis miR-145.
 54. A method of claim 49, which further comprises: comparingthe level of at least one additional miR gene product in a test samplefrom the subject to a control level of the miR gene product, wherein theat least one additional miR gene product is selected from the groupconsisting of: miR-21; miR-29b-2; miR-146; miR-125b-2; miR-125b-1;miR-10b; miR-145; miR-181a; miR-140; miR-213; miR-29a prec; miR-181b-1;miR-199b; miR-29b-1; miR-130a; miR-155; let-7a-2; miR-205; miR-29c;miR-224; miR-100; miR-31; miR-30c; miR-17-5p; miR-210; miR-122a; andmiR-16-2.
 55. A method of diagnosing whether a subject has, or is atrisk for developing, breast cancer, comprising: a.) comparing the levelof at least miR-146 gene product and at least one additional miR geneproduct in a test sample from a subject to a control level of at leastone corresponding miR-146 gene product and at least one additional miRgene product; and b.) diagnosing whether the subject has, or is at riskfor developing, breast cancer, if the level of the at least one miR-146gene product and at least one additional miR gene product in the testsample from the subject is greater than a control level of miR-146 geneproduct and less than a control level of at least one correspondingadditional miR gene product.
 56. A method of claim 55, wherein the atleast one additional miR gene product is miR-125b-2.
 57. A method ofclaim 55, wherein the at least one additional miR gene product ismiR-125b-1.
 58. A method of claim 55, wherein the at least oneadditional miR gene product is miR-10b.
 59. A method of claim 55,wherein the at least one additional miR gene product is miR-145.
 60. Amethod of claim 55, which further comprises: comparing the level of atleast one additional miR gene product in a test sample from the subjectto a control level of the miR gene product, wherein the at least oneadditional miR gene product is selected from the group consisting of:miR-21; miR-29b-2; miR-146; miR-125b-2; miR-125b-1; miR-10b; miR-145;miR-181a; miR-140; miR-213; miR-29a prec; miR-181b-1; miR-199b;miR-29b-1; miR-130a; miR-155; let-7a-2; miR-205; miR-29c; miR-224;miR-100; miR-31; miR-30c; miR-17-5p; miR-210; miR-122a; and miR-16-2.61. A method of diagnosing whether a subject has, or is at risk fordeveloping, breast cancer, comprising: a.) reverse transcribing at leasttwo miR gene products from a test sample from the subject to provide atleast two corresponding miR gene product target oligonucleotides; b.)hybridizing at least two miR target oligodeoxynucleotides to amicroarray comprising miRNA-specific probe oligonucleotides that includeat least two corresponding miRNA-specific probe oligonucleotides toprovide a hybridization profile for the test sample; c.) comparing thetest sample hybridization profile to a control hybridization profile forat least the two miR gene products; and d.) diagnosing whether thesubject has, or is at risk for developing, breast cancer if the level ofat least two gene products in the test sample is greater than thecontrol level of the two gene products, wherein the two miR geneproducts are gene products of the miRs selected from the groupconsisting of: miR-21; miR-29b-2; miR-146; miR-125b-2; miR-125b-1;miR-10b; miR-145; miR-181a; miR-140; miR-213; miR-29a prec; miR-181b-1;miR-199b; miR-29b-1; miR-130a; miR-155; let-7a-2; miR-205; miR-29c;miR-224; miR-100; miR-31; miR-30c; miR-17-5p; miR-210; miR-122a; andmiR-16-2.